3rd Wroclaw Scientific Meetings

3rd Wroclaw Scientific Meetings

Editors: Julita Kulbacka Nina Rembiałkowska Joanna Weżgowiec

Wroclaw 2019

3rd Wroclaw Scientific Meetings Wrocław, 1st-2nd March 2019

Editors: Julita Kulbacka Nina Rembiałkowska Joanna Weżgowiec

Typesetting: Monika Maciąg Kamil Maciąg

Cover design: Marcin Szklarczyk Nina Rembiałkowska

© Copyright by Wydawnictwo Naukowe TYGIEL Sp. z o.o.

ISBN 978-83-65932-64-8

Publisher: Wydawnictwo Naukowe Tygiel Sp. z o.o. ul. Głowackiego 35/341, 20-060 Lublin www.wydawnictwo-tygiel.pl

Scientific Committe: prof. Janina Kwiatkowska-Korczak Department of Medical Biochemistry, Wroclaw Medical University, Poland prof. Halina Grajeta Department of Food Science and Dietetics, Wroclaw Medical University, Poland prof. dr hab. Izabela Fecka Department of Pharmacognosy and Herbal Medicines, Wroclaw Medical University, Poland prof. Halina Milnerowicz Department of Biomedical and Environmental Analyses, Wroclaw Medical University, Poland prof. Bożena Karolewicz Department of Drugs Form Technology, Wroclaw Medical University, Poland prof. Mounir Tarek Centre National de La Recherche Scientifique (CNRS) Université de Lorraine, Nancy, France prof. Saulius Šatkauskas Department of Biology, Vytautas Magnus University in Kaunas, Lithuania prof. Marcin Drąg Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Science and Technology Biochemistry, Wroclaw, Poland prof. dr hab. Agnieszka Piwowar Department of Toxicology, Wroclaw Medical University, Poland dr hab. Jolanta Saczko, prof. nadzw. Department of Molecular and Cellular Biology, Wroclaw Medical University, Poland dr hab. inż. Julita Kulbacka Department of Molecular and Cellular Biology, Wroclaw Medical University, Poland dr hab. Iwona Bil-Lula Department of Medical Laboratory Diagnostics, Wroclaw Medical University, Poland

dr Agnieszka Chwiłkowska Department of Molecular and Cellular Biology, Wroclaw Medical University, Poland dr Helena Moreira Department of Basic Medical Sciences, Wroclaw Medical University, Poland dr inż. Nina Rembiałkowska Department of Molecular and Cellular Biology, Wroclaw Medical University, Poland dr inż. Dagmara Baczyńska Department of Molecular and Cellular Biology, Wroclaw Medical University, Poland dr inż. Anna Choromańska Department of Medical Biochemistry, Wroclaw Medical University, Poland dr inż. Joanna Rossowska Institute of Immunology and Experimental Therapy, Poland dr Vitalij Novickij Institute of High Magnetic Fields, Vilnius Gediminas Technical University, Lithuania dr Małgorzata Drąg-Zalesińska Department of Human Morphology and Embryology, Wroclaw Medical University, Poland

Organizing Committe: dr hab. Jolanta Saczko, prof. nadzw. Department of Medical Biochemistry, Wroclaw Medical University, Poland prof. dr hab. Agnieszka Piwowar Department of Toxicology, Wroclaw Medical University, Poland prof. dr. hab. Adam Matkowski Department of Biology and Pharmaceutical Botany, Wroclaw Medical University, Poland dr hab. inż. Julita Kulbacka Department of Medical Biochemistry, Wroclaw Medical University, Poland

dr hab. Ewa M. Kratz Department of Laboratory Diagnostics,Wroclaw Medical University, Poland dr inż. Nina Rembiałkowska Department of Medical Biochemistry, Wroclaw Medical University, Poland dr inż. Dagmara Baczyńska Department of Molecular and Cellular Biology, Wroclaw Medical University, Poland dr Agnieszka Chwiłkowska Department of Medical Biochemistry, Wroclaw Medical University, Poland dr inż. Anna Choromańska Department of Medical Biochemistry, Wroclaw Medical University, Poland dr Helena Moreira Department of Basic Medical Sciences, Wroclaw Medical University, Poland dr Ewa Sawicka Department of Toxicology, Wroclaw Medical University, Poland dr inż. Joanna Weżgowiec Department of Experimental Dentistry, Wroclaw Medical University, Poland dr inż. Joanna Rossowska Institute of Immunology and Experimental Therapy, Poland dr Anna Szewczyk Department of Molecular and Cellular Biology, Wroclaw Medical University, Poland dr Adriana Kubis-Kubiak Department of Toxicology, Wroclaw Medical University, Poland dr Beata Szymańska Department of Toxicology, Wroclaw Medical University, Poland mgr Olga Michel Department of Medical Biochemistry, Wroclaw Medical University, Poland

mgr Katarzyna Bieżuńska-Kusiak Department of Medical Biochemistry, Wroclaw Medical University, Poland Karolina Tądel Wroclaw Medical University, Poland Marek Kulbacki DIVE IN AI, Polish Japanese Academy of Information Technology, Poland

Orgnizers:

Honorary Patronage:

Sponsors:

Content:

CONFERENCE PROGRAMM ...... 15

LECTURES ...... 19

ORAL PRESENTATIONS ...... 27

POSTER PRESENTATIONS ...... 53

INDEX OF AUTHORS ...... 187

Dear Sir or Madam,

This is the third scientific meeting in Wroclaw. This year the Department of Molecular and Cellular Biology together with the Department of Toxicology has already organized the conference entitled "3rd Wroclaw Scientific Meetings", dedicated to young scientists. According to tradition, the aim of the conference is to exchange information about scientific research between young people, enthusiasts of science from many centers in Poland and abroad. The conference is multidisciplinary and covers broadly understood cell biology in the field of drug interactions in cancer and normal cells, cell-drug interaction, cellular response to oxidative stress, cellular engineering, including computer modeling. During the conference, young scientists will have the opportunity to present the results of their research and discuss them with experienced and valued people of science, who were invited to participate in this year's conference. I would like to emphasize that this year's plenary lectures will be presented by world-renowned scientists from Poland and abroad. During the conference, practical workshops on the flow cytometry will also be organized. I cordially invite you to take part in them. I wish all conference participants fruitful discussions and nice moments in Wrocław. We are very happy that our conference meets with growing interest of young science enthusiasts not only from Poland but also from foreign centers.

Dr hab. Jolanta Saczko, prof. nadzw.

CONFERENCE PROGRAMM

01.03.2019 Friday 08:00-10:00 Registration 10:15-10:30 Opening ceremony – Prof. Jolanta Saczko and Prof. Halina Grajeta

1st SESSION Moderators: Marcin Drąg and Jolanta Saczko 10:30-11:00 Lecture I of Prof. Marcin Drąg (Wroclaw University of Science and Technology, Wroclaw, Poland) “In Quest of Optimal Technology to Obtain Selective Activity-Based Probes for Investigation of Proteases in Cell Death” 11:00-11:12 Dominika Benkowska “Lyotropic liquid crystals in biological systems” 11:13-11:24 Klaudia Bielak “Otolin-1 – expression and purification of the structural protein of fish otoliths and vertebrate otoconia” 11:25-11:36 Ewa Kątnik “The role of BCL11A protein in non-small cell lung cancer” 11:37-11:48 Krzysztof Kotowski “PFKFB3 inhibition by use of 3PO induce apoptosis and cell cycle arrest in A375 human melanoma cell Line” 11:49-12:00 Marta Piksa “Photosensitizers as anti-inflammatory agents in the diabetic foot” 12:00-12:30 Coffee break and POSTER SESSION

15 2nd SESSION Moderators: Joanna Rossowska and Helena Moreira 12:30-13:00 Lecture II of Dr. Joanna Rossowska (Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland) “Flow cytometry applications in medical and biological sciences” 13:00-13:15 Dawid Lupa “Noble metal nanoparticles monolayers at colloidal carriers - novel systems for controlled release” 13:15-13:30 Matylda Niedziela “Influence of Pelargonium sidoides proanthocyanidins on metabolism and apoptosis of Dental Pulp Stem Cells” 13:30-13:45 Magdalena Surman “Comparison of surface glycosylation of melanoma cells and melanoma-derived ectosomes” 13:45-14:00 Dries Van Elst “Mechanisms and novel treatment approaches in allergen immunotherapy” 14:00-15:30 Lunch and POSTER SESSION

14:15-15:45 Flow Cytometry Workshops (Two groups 14:15-15:00, II – 15:00-15:45)

3rd SESSION Moderators: Mounir Tarek and Łukasz Radosiński 15:45-16:15 Lecture III of Prof. Mounir Tarek (CNRS- University de Lorraine, Nancy, France) “Drug delivery through cell membranes: What can we learn from Molecular Dynamics simulations” 16:15-16:27 Łukasz Lewandowski “Organs-on-a-chip: an innovative tool in toxicological studies” 16:28-16:39 Wojciech Szlasa “How to predict the secondary from the primary structure of the protein?”

16 16:40-16:51 Natalia Szulc “Calcium electroporation- a molecular dynamics simulation” 16:52-17:03 Sebastian Szymański “Hypericin and phenothiazines – the use of computational methods to determine the structure and properties of drugs” 17:04-17:15 Daniel Wiczew “Pro-oxidative properties of cynamonic acid towards normal and cancer human cells” 17:15-17:45 POSTER SESSION – diner & discussions

02.03.2019 Saturday

4th SESSION Moderators: Saulius Šatkauskas and Julita Kulbacka 9:00-9:30 Lecture IV of Prof. Saulius Šatkauskas (Vytautas Magnus University, Kaunas, Lithuania) “Cell sonoporation: from basics to targeted drug delivery” 9:30-9:42 Ludmila Bogacz-Radomska “Carotenoids as Source of Antioxidant Acitivity in Food Matrix” 9:43-09:54 Justyna Kiszkiel “Synthesis and amidolytic activity of fragment of lunasin” 09:55-10:06 Paulina Komorek “Changes In Lysozyme’s II-Structure As A Result Of Its Interaction With A Gold Surface – Preferred Conditions for Lysozyme’s Aggregates Formation in Neurodegenerative Disease Development” 10:07-10:18 Stanislaw Kwiatkowski “Anti-cancer effect of photodynamic therapy and electroporation mediated by curcumin on the A375 melanoma cell line” 10:19-10:30 Alicja Surowiak “Oximes derived from natural volatile compounds as antimicrobial active agents” 10:30-11:00 Coffee break and POSTER SESSION

17 5th SESSION Moderators: Ian Azarov and Agnieszka Piwowar 11:30-12:00 Lecture V of Dr Ian Azarov (Ural Branch of Russian Academy of Sciences, Syktyvkar, Komi Republic, Russia) “Melatonin as an antiarrhythmic agent – electrophysiological targets in the ischemic myocardium” 12:00-12:12 Jagoda Karczewska “Impact of demographic factors on prevalence, severity level and course of urticaria in patients in Department of Allergy, Clinical Immunology and Internal Medicine in 2012-2018 years” 12:13-12:24 Marta Banaszkiewicz “An association between MMP-2, MMP-9, TIMP-4 and stable coronary artery disease, hypertension and diabetes” 12:25-13:36 Gabriela Bis “Graft-versus-host disease in posttrans-plantational pediatric patients” 13:37-13:48 Kamila Kołodziejczyk “Studies of haemostatic dressings reaction on fibroblasts and microorganisms” 13:49-13:00 Piotr Kuropka “The use of thermography in experimentally induced pleurisy induced by low dose of TCDD” 13:00-13:30 Coffee break

13:30-13:45 Announcement of the results for the best presentation and closing ceremony, certificates.

18

LECTURES

L 1. In Quest of Optimal Technology to Obtain Selective Activity-BasedProbes for Investigation of Proteasesin Cell Death Marcin Drąg1,2 1Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Science and Technology, Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland 2Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA Proteolysis is one of the most important and ancient reactions in biology. Enzymes that catalyze this reaction are called proteases. Proteases as “good guys” perform many significant biological processes like cellular quality control, apoptosis, blood coagulation or signal transduction. However they can be also “bad guys” contributing to pathological events like cancer, diabetes, coagulopathies, inflammation, infectious or degenerative diseases. It is estimated that 5-10% of all pharmaceutical targets being pursued for drug development are proteases. Despite significant progress in recent years, one of the biggest problems in the investigation of proteases is their similar activity and location. Due to the overlapping substrate specificity (preference in the recognition of natural amino acids) it is very hard to distinguish many major proteolytic enzymes families using chemical tools developed using classic screening technologies. This also very often limits discovery of selective drug or marker for specific activity monitoring. Classic examples here are cysteine proteases from caspase family, which play crucial role in controlling of several major metabolic pathways, especially apoptosis. Our group has recently developed technology to obtain new types of ultrasensitive chemical tools (substrates, inhibitors, activity-based probes) for major families of medically important proteases. Using this novel, unique and very efficient technology called Hybrid Combinatorial Substrate Library (HyCoSuL) we have demonstrated that protease substrate specificity can be significantly enlarged by the use of unnatural amino acids in peptide sequence. An overview of major strategies to develop very active and selective chemical tools, which can be used for reliable investigation of activity and location of proteases in health and disease will be presented. REFERENCES: 1. Drag & Salvesen, Nature Reviews Drug Discovery 2010, 9, 690. 2. Kasperkiewicz et al., FEBS Journal 2017, 284, 1518. 3. Kasperkiewicz et al., Journal of the American Chemical Society 2017, 139, 10115. 4. Poreba et al., Nature Protocols, 2017, 12(10), 2189. 5. Poreba et al., Chemical Science, 2018, 9(8), 2113. 6. Rut et al., J. Med. Chem., 2018, 61(12), 5222.

Prof. Marcin Drąg is a professor at Wrocław University of Science and Technology (Poland) & Professor Adjunct at SBP Medical Discovery Institute (USA).

21 L 2. Flow cytometry applications in medical and biological sciences Joanna Rossowska

Hirszfeld Institute of Immunology and Experimental Therapy, Wrocław, Poland The flow cytometry method is a reliable and increasingly manageable tool for the quantitative analysis of cells or particles. Modern flow cytometers are able to analyze many thousand particles per second, and, if configured as cell sorters, can actively separate and isolate particles with specified optical properties. Increasing number of lasers and detectors in the cytometers allows for multiple antibody labeling, and more precise identification of target population by their phenotypic markers. Thus, it allows the measurement of many different parameters including expression of surface or intracellular antigens, apoptosis, DNA damage, or cell cycle. In view of the above this technology can be applied in a number of fields, including molecular biology, pathology, immunology, virology or plant biology. It is broadly applied in medicine especially in transplantation, hematology or tumor immunology. Flow cytometry also is feasible for difficult tissues such as tumors. In our research we use the multicolor flow cytometry for detailed quantitative analysis of tumor microenvironment heterogeneity. The aim of our research is to determine the changes ongoing in TME after application of dendritic cell-based therapy supported by immunomodulating factors like inhibitors of immunosuppressive TGF-β1 or IL-10. Advanced multicolor cytometry gives us the possibility for simultaneous analysis of a number as well as functional and phenotypical changes of immune cells infiltrating tumors. Based on our recent investigation, the role of flow cytometry in tumor diagnosis and prevention as well as the advantages and limitations of the technique will be discussed.

Dr Joanna Rossowska is an assistant professor at Hirszfeld Institute of Immunology and Experimental Therapy (Poland). Her research focuses on the development of new anticancer immunotherapies which aim is to reactivate potent host antitumor response through reprogramming the hostile tumor environment.

22 L 3. Drug delivery through cell membranes: What can we learn from Molecular Dynamics simulations Mounir Tarek

Centre National de La Recherche Scientifique (CNRS) Université de Lorraine, Nancy FRANCE Membranes constitute an important component of cells. Not only they give support for trans- membrane proteins to anchor in, but act also as natural barriers against free translocation of molecules and nutrients as they prevents them from going in or out of the cell. Such is the case for many ions, charged compounds and large polar molecules, e.g., drugs that in order to be effective need to cross these barriers. Different strategies have been developed during the past decades to transfer molecules of variable sizes and chemical properties across membranes of a cell. These include the use of nanomaterials such as carbon nanotubes and fullerenes as carriers, or enhancement of membranes permeability by electroporation. Regardless of the method used to vectorize small fragments or molecules inside cell compartments, understanding their interaction with biological membranes and their behavior while they penetrate their hydrophobic core is of crucial importance. The direct observation of such events is not possible with conventional techniques. Furthermore, due to the complexity and heterogeneity of cell membranes, it is difficult to describe and characterize their behavior in terms of atomically resolved processes. Atomistic simulations in general and Molecular Dynamics (MD) simulations in particular, have proven to be effective for providing insights into both the structure and the dynamics of model lipid membranes in general. Here we provide a review of the progress made so far in modeling transport phenomena across such membranes. We show how such “computer experiments” provide significant insights into the transport of a wide variety of components. We discuss in particular, based on our recent investigations how transport of species ranging from small amino acids to large molecules occurs by direct translocation, and how it might be enhanced by anchoring them to nanomaterials or by subjecting the systems to high electric fields.

Prof. Mounir Tarek is a Senior Research Director at the CNRS-Université de Lorraine, recipient of a Ph.D. in Physics from the University of Paris in 1994. He joined the CNRS after a four-years tenure at the National Institute of Standards and Technology (Gaithersburg Maryland USA) following three years tenure at the University of Pennsylvania). His research focus and expertise lies in the study of cell membranes transport processes. It involves the use of computational chemistry methods to study membranes, proteins, ion channels and membrane transport proteins. Over the last few years, he studied many aspects of electroporation of cell membranes subject to high electric fields.

23 L 4. Cell sonoporation: from basics to targeted drug delivery Saulius Šatkauskas

Department of Biology, Vytautas Magnus University in Kaunas (Lithuania) Cell sonoporation is a novel therapeutic technique used for noninvasive drug and gene transfer to cells and tissues. Sonoporation can be induced during cell exposure to ultrasound (US) in the presence of an US contrast agent microbubbles (MB). The mechanism of cell sonoporation is not known in detail, however crucial role plays US-induced MB cavitation. Cavitating MB induce microstreaming and microjets that creates shear forces acting on the cell membranes. MB cavitation also results in the generation of reactive oxygen species and the formation of free radicals, therefore producing sonochemical effects on membranes, as well as in an increase of the local temperature that enhances the fluidity of the membranes. It has been shown that sonoporation can be used as an effective method for intracellular transfer of anticancer drugs, like bleomycin, camptomycin, mitoxantrone, and doxorubicin, used either for cytotoxicity increase in vitro or antitumor therapy in vivo. In addition, MB can be loaded with various agents and therefore can be exploited as drug carriers. The potential use of MB and sonoporation for clinical application will be discussed.

Prof. Saulius Šatkauskas is a professor in Department of Biology, Vytautas Magnus University in Kaunas (Lithuania). He is also head of Biophysical Research Group. His research lies in the fields of membrane electropermeabilization and sonoporation in cells and tissues.

24 L 5. Melatonin as an antiarrhythmic agent – electrophysiological targets in the ischemic myocardium Jan Azarov

Institute of Physiology, Komi Science Centre, Ural Branch of Russian Academy of Sciences, Syktyvkar, Komi Republic, RUSSIA Ventricular tachyarrhythmias, especially fibrillation, are frequent and often fatal complications of acute myocardial infarction. Development of cardioprotective drugs with antiarrhythmic properties presents an important research challenge. Melatonin is positioned as a promising cardioprotective medication, whose effects are thought to be largely mediated by its antioxidative properties. On the other hand, a role of signaling pathways via melatonin receptors in the cardioprotective and specifically antiarrhythmic effects of melatonin still need to be determined. As ventricular fibrillation is primarily an electrophysiological phenomenon, it is reasonable to attempt managing this arrhythmia on the basis of electrocardiographic predictions and targeting at specific electrophysiological substrates. Melatonin and some other antioxidants provide pronounced myocardial electrophysiological effects in the ischemic conditions. Dispersion of repolarization, a prerequisite of reentrant arrhythmias, was significantly reduced by several antioxidants. However, few of them had documented antiarrhythmic effects. On the other hand, melatonin not only modified the repolarization parameters but also improved activation spread acting on cellular and tissue levels. The melatonin effect on activation was associated with reduction of ventricular fibrillation incidence, did not relate to oxidative stress but was abolished by the block of MT1/MT2 receptor. These data suggest involvement of MT1/MT2 signaling pathway in the antiarrhythmic action of melatonin.

Jan Azarov is a Chief Researcher at the Department of Cardiac Physiology, Institute of Physiology, Komi Science Centre of Ural Branch of Russian Academy of Sciences and a Professor at the Department of Physiology, Pitirim Sorokin Syktyvkar State University (Syktyvkar, Komi Republic, Russia). His research is concerned with myocardial electrical heterogeneity, its mechanism, ECG expression and functional significance – specifically, a role it plays in arrhythmogenesis.

25

ORAL PRESENTATIONS

1st SESSION O 1. Lyotropic liquid crystals in biological systems D. Benkowska, K. Matczyszyn

Advanced Materials Engineering and Modelling Group, Wroclaw University of Science and Technology, Poland Liquid crystals (LCs) are state of matter possessing prosperities between liquid and three dimensionally ordered solid. These structures are distinguished in the thermotropic and lyotropic phase due to causes for their phase transition. In biological material, where samples need to be fluid and crystalline with respect to concentration, structures show lyotropic arrangement. One of the promising examples of LCs in biology is DNA, which at high concentration tends to self- organize into lyotropic mesophases. Furthermore, it was shown that the idea of doping DNA LC matrix with gold nanoparticles (NPs) could be essential in order to use them in biomedical applications such as bioimaging or plasmonics-enhanced cancer thermotherapy [1-3]. In this study, we present the formation of DNA LCs doped with elongated gold nanoparticles and compare them with pure DNA LCs. Our goal is to characterize and understand the impact of gold nanostructures onto DNA LCs formation and manipulation of the obtained systems with temperature and/or light. Various textures of DNA mesophases and the temperature of phase transitions were investigated by polarized light microscopy equipped with the hot-stage.

Fig. 1 (a) Cholesteric texture of DNA liquid crystals. Scale bar: 50µm. (b) Columnar hexagonal phase in DNA liquid crystals. Scale bar: 100µm. REFERENCES: 1. J. Olesiak-Banska, M. Gordel, K. Matczyszyn, V. Shynkar, J. Zyss, M. Samoc, Nanoscale, 5, 10975- 10981, 2013. 2. K. Brach, M. Waszkielewicz, J. Olesiak-Banska, M. Samoc, K. Matczyszyn, Langmuir, 33, 36, 8993- 8999, 2017. 3. K. Brach, K. Matczyszyn, J. Olesiak-Banska, M.Gordel, M. Samoc, Phys. Chem. Chem. Phys., 18, 7278-7283, 2016.

29 O 2. Otolin-1 – expression and purification of the structural protein of fish otoliths and vertebrate otoconia K. Bielak1, A. Zoglowek1, P. Dobryszycki1

1Department of Biochemistry, Wrocław University of Science and Technology, Wrocław, Poland Biologically controlled mineralization, also referred as biomineralization, is a process in which living organisms create minerals significant for their proper functioning as inorganic parts of skeletons, shells and other hard tissues [1]. Mineralization in living organisms is in control of distinct proteins, which modulate the crystal growth and support the deposition of minerals on the organic scaffold [1]. One of the examples of mineralized structures are fish otoliths (ear stones) and vertebrate otoconia (ear dust), composed of calcium carbonate crystals placed on the organic matrix [2]. One of the most abundant organic element of the scaffold is a collagenous protein named otolin-1 containing C-terminal globular C1q domain [3]. Here we present preliminary purification protocol for recombinant otolin-1 from Danio rerio. The expression system was composed of E.coli cells transformed with a vector containing otolin-1 cDNA that was optimized for the expression in prokaryotic organisms. The protein was purified with chromatographic methods, namely metal affinity chromatography and size exclusion chromatography. ACKNOWLEDGEMENTS The authors thank dr inż. Rafał Hołubowicz for providing the cDNA of otolin-1 from Danio rerio. REFERENCES: 1. R. Hołubowicz, et al. “Biomineralizacja – kontrolowana przez białka precyzja kształtu, struktury I właściwości”, Postępy Biochemii 61.4, 2015. 2. Y. W. Lundberg, et al. “Mechanisms of otoconia and otolith development”, Developmental Dynamics 244.3, 2015. 3. E. Murayama, et al. “Fish otolith contains a unique structural protein, otolin‐1”, European journal of biochemistry 269.2, 2002.

30 O 3. The role of BCL11A protein in non-small cell lung cancer

E. Kątnik1, A. Piotrowska1, J. Grzegrzółka1, A. Gomułkiewicz1, P. Dzięgiel1 1Department of Histology and Embryology, Wroclaw Medical Uniwersity, Wroclaw, Poland Lung cancer, despite significant advances in diagnosis and treatment, is still one of the most common malignant tumours worldwide. The identification of new diagnostic markers is still very important to reduce patients mortality due to this tumour. One of such markers may be the BCL11A protein. It is a transcription factor regulating the expression of genes involved in numerous intracellular processes, e.g. apoptosis and cell cycle. Increased expression of BCL11A has been demonstrated in some tumours, including non-small cell lung cancer (NSCLC). However the mechanisms underlying the action of BCL11A have not been clarified yet. Thus, we aimed at determining its role in processes associated with NSCLC progression. In the study, we evaluated the relationship between the level of BCL11A and proliferation antigen (Ki-67) and epithelial-mesenchymal transition (EMT) markers (Slug, Snail). The study used paraffin blocks of 250 NSCLC cases (SCC – squamous cell carcinoma and AC – adenocarcinoma) and 110 sections of noncancerous tissue. The levels of BCL11A, Ki-67, Slug and Snail were determined by immunohistochemistry on tissue microarrays. The semi- quantitative IRS scale was used to assess the immunohistochemical reactions. Statistical analysis was performed using the Statistica 13.1. In SCC, the nuclear expression of BCL11A was observed, whereas in AC in the cytoplasm of tumour cells. In SCC, the mean positive correlation between BCL11A and Ki-67 was reported. Moreover, the positive correlation was observed between BCL11A and the level of both investigated EMT markers, Slug and Snail. The results suggest that BCL11A may be involved in the NSCLC progression and metastasis.

31 O 4. PFKFB3 inhibition by use of 3PO induce apoptosis and cell cycle arrest in A375 human melanoma cell line K. Kotowski1, D. Przystupski1, S. Kwiatkowski1, A. Kielbik1, P. Wawryka1, O. Michel2, J. Saczko3, J. Kulbacka3 1Faculty of Medicine, Wroclaw Medical University, Wroclaw, Poland 2Department of Medical Biochemistry, Wroclaw Medical University, Wroclaw, Poland 3Department of Molecular and Cellular Biology, Wroclaw Medical University, Wroclaw, Poland The global incidence of melanoma tends to rise, reaching in Poland almost 3-fold in past 30 years. Around 50% of melanoma cases are BRAF-positive tumors, in this group more than 90% are BRAFV600E positive. The alteration in the gene leads to consistent and potent activity of Braf kinase in pathway RAS-RAF-MEK-ERK. As a result the abnormality promotes stabilization of HIF-1α, the promoter of PFKFB3. The application of the inhibitors might be effective but it also has some severe side effects such as cardiotoxicity, secondary cancers and acquired resistance for Braf inhibitors. Thus, in this research we decided to investigate intriguing alternative agent (2E)-3-(3-Pyridinyl)-1-(4- pyridinyl)-2-propen-1-one (3PO) - small molecule selective inhibitor of PFKFB3. The purpose of the study was to verify the effect of 3PO on BRAFV600E positive human melanoma cells (A375), as an alternative to vemurafenib-based treatment. The MTT viability assay was used to estimate the promotion of cytotoxic effect of 3PO agent. The type of cell death was determined by Annexin V (FITC) and PI method by flow cytometry. Immunocytochemical staining was carried out following the ABC protocol using caspase-3 and -8 antibodies. Additionally, the cell cycle arrest induction by 3PO was performed according to the cell cycle profiling using FACS analysis (propidium iodide statining). The results show that 3PO display a dose-dependent, but time-independent, cytotoxic effect on A375 melanoma cells. The death of cells occurred mostly through the early apoptosis. Moreover the results indicate that 3PO may induce cell cycle arrest in G2/M phase. ACKNOWLEDGEMENTS The research was supported by the Scientific Cancer Cell Biology Group No. 148 (WMU), Polish Science Ministry grant ,,Najlepsi z Najlepszych 3.0!”, Statutory Funds of Department of Molecular and Cellular Biology and was partially performed in the Screening Laboratory of Biological Activity Test and Collection of Biological Material, Faculty of Pharmacy and the Division of Laboratory Diagnostics, Wroclaw Medical University, supported by the ERDF Project within the Innovation Economy Operational Programme POIG.02.01.00-14-122/09

32 O 5. Photosensitizers as anti-inflammatory agents in the diabetic foot

M. Piksa1, C. Lian2, K. Yoshida2, S. Persheyev2, K. Pawlik3, K. Matczyszyn1, I. D. W. Samuel2

1 Advanced Materials Engineering and Modelling Group, Faculty of Chemistry, Wroclaw University of Science and Technology, Wrocław, Poland 2 Organic Semiconductor Centre, School of Physics and Astronomy, University of St Andrews, St Andrews, UK 3 Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland The dramatic increasing occurrence of antibiotic resistance bacteria has become nowadays one of the biggest threats to global public health. When antibiotic treatment failure, it is necessary to investigate new potential antimicrobial therapeutic modality. A promising alternative to antibiotic is antimicrobial photodynamic therapy (aPDT) bases on photosensitizers activated by light and capable by absorbing energy to produce ROS - Reactive Oxygen Species[1–3]. A major research topic is the most commonest bacterial cultures of diabetic foot. Currently we are focused on the opportunistic bacteria Staphylococcus aureus, which is the reason for majority health care–associated infections and the essential infection agents of this chronic complication of diabetes mellitus. We had investigate the influence of activated methylene blue as a photosen- sitizer. This compound by irradiation generate primarily singlet oxygen considered to be major antibacterial agent [4,5]. Flexible OLED (organic light-emitting diode) is used as a PDT light source. This low driving voltage device is tuneable whereby it is able to get optimal wavelength which effective activate photosensitizer but it is also safe for bacteria and does not destroy their cells. The results show a significant bacterial load reduction by light-activated photosensitizers and killing rate depends on irradiation- time and can amount to even approx. 99%. REFERENCES: 1. Alasqah MN. Antimicrobial efficacy of photodynamic therapy on dental implant surfaces: A systematic review of in vitro studies. Photodiagnosis Photodyn Ther 2019; 25: 349-53. doi:10.1016/j.pdpdt.2019.01.018. 2. Huang T-C, Chen C-J, Ding S-J, Chen C-C. Antimicrobial efficacy of methylene blue-mediated photodynamic therapy on titanium alloy surfaces in vitro. Photodiagnosis Photodyn Ther 2019; 25: 7-16. doi:10.1016/J.PDPDT.2018.11.008. 3. MULTIDRUG-RESISTANT TUBERCULOSIS (MDR-TB). 2017. 4. Klevens RM, Morrison MA, Nadle J, Petit S, Gershman K, Ray S, et al. Invasive Methicillin-Resistant <EMPH TYPE="ITAL">Staphylococcus aureus</EMPH> Infections in the United States. JAMA 2007;298:1763. doi:10.1001/jama.298.15.1763. 5. Tenover FC. Mechanisms of antimicrobial resistance in bacteria. Am J Infect Control 2006; 34: S3-10. doi:10.1016/J.AJIC.2006.05.219.

33 2nd SESSION O 6. Noble metal nanoparticles monolayers at colloidal carriers – novel systems for controlled release D. Lupa, M. Oćwieja, Z. Adamczyk

Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Krakow, Poland Controlled release of active substances (AA) from colloidal vehicles is one of the most promising strategies for targeted drug delivery. So far, the various AA release profiles under various conditions were quantitatively evaluated, whereas the mechanisms of these processes was not systematically investigated. Taking into account that detailed understanding of those phenomena is key factor in developing of novel targeted therapies, the main goal of this work is to investigate the mechanism of nanoparticle release from monolayers formed at colloidal carriers - a novel type of system with potential use as delivery substrates. Due to their biocompatibility, positively-charged gold nanoparticles (AuNP) [1] and negatively-charged silica microparticles (SMP) [2] were chosen as substrates for investigated system. After purification of native AuNP and SMP suspensions, their basic physicochemical properties (hydrodynamic diameter, electrokinetic charge) were determined under broad range of pHs and ionic strength values. In the next step, AuNPs were immobilized on SMP in colloidal self-assembly process [3]. After completion of AuNPs deposition, the physicochemical properties of AuNPs@SMP microcomposites were determined. Then, the changes in AuNPs coverage over time were evaluated using various techniques: electrokinetic measurements, SEM method and the indirect colloidal deposition method. Comparison of experimental results with theoretical calculations revealed that electrostatic interaction energy is the decisive parameter determining the stability of nanoparticle monolayers. This important finding implies that AuNPs release can be regulated by change in external parameters such as pH and ionic strength. ACKNOWLEDGEMENTS The authors gratefully acknowledge the financial support of the National Science Centre (OPUS Project UMO-2015/19/B/ST5/00847) REFERENCES: 1. R. Shukla et al. Langmuir vol. 21 pp. 10644-10654, 2005. 2. T. Asefa et al. Chem. Res. Toxicol. vol. 25 pp. 2265-2284, 2012. 3. D. Lupa et al. Colloids Surf. A vol. 554 pp. 317-325, 2018.

34 O 7. Influence of Pelargonium sidoides proanthocyanidins on metabolism and apoptosis of Dental Pulp Stem Cells M. Niedziela1, P. Ceglarek1, T. Męcik-Kronenberg2, A. M. Osyczka1, G. Tylko1

1Department of Cell Biology and Imaging, Institute of Zoology and Biomedical Researches, Jagiellonian University, Kraków, Poland 2Department of Pathomorphology, Medical University of Silesia, Katowice, Poland Proanthocyanidins (PACS) are plant-derived flavonoids. PACS have been used as pigments for many years but due to their antioxidant, anticancer, antimicrobial, and anti-inflammatory pro- perties, PACS might be used in many therapies, cancer treatments or regenerative medicine [1]. Teeth are excellent source of stem cells applicable in regeneration of many tissues. They are easy to obtain and might successfully replace bone marrow stem cells [2]. The aim of the study was to establish in vitro effects of P. sidoides PACS on tooth-derived stem cells. Dental Pulp Stem Cells (DPSC) were obtained from healthy third molars and cultured in Complete Growth Medium (CGM) [3]. Next, the population was divided into three experimental groups: I) cells cultured in CGM (Control), II) cells treated for 24h, 48h, and 72h with 0.01% PACS, or III) 0.1% PACS dissolved in CGM. Mitochondrial membrane potential (MMP) and annexin-V/propidium iodide exposure were determined by flow cytometry with concomitant analysis of caspase-8 and caspase-9 activities. The number of cells with low MMP increased significantly after 24h, 48h and 72h treatment with 0.01% of PACS when compared to the Control. However, no apoptotic features were observed in DPSCs when treated with both concentrations of PACS. Since caspase-8 activity of DPSCs remained unchanged, significant decrease of caspase-9 was determined in DPSCs treated with 0.1% PACS. Concluding, P. sidoides proanthocyanidins change DPSCs metabolism, however, this effect did not contribute to apoptosis of the cells. It rather suggests that PCAN impairs apoptosis associated with mitochondria by lowering the activity of caspase-9. ACKNOWLEDGEMENTS The studies supported by M.ERA-NET2 PELARGODONT grant (NCN-2016/22/Z/ST500692). REFERENCES: 1. T. Brendler, B. Van Wyk, Journal of Ethnopharmacology, vol. 119 p. 420-433, 2008. 2. P. T. Sharpe, Development, vol. 143 p. 2273-2280, 2016. 3. P. Di Nardo, et al., Methods in Molecular Biology, vol. 1553 p. 191-207, 2017.

35 O 8. Comparison of surface glycosylation of melanoma cells and melanoma – derived ectosomes M. Surman1, A. Drożdż2, E. Stępień2, M. Przybyło1

1Department of Glycoconjugate Biochemistry, Institute of Zoology and Biomedical Research, Jagiellonian University, Kraków, Poland 2Department of Medical Physics, M. Smoluchowski Institute of Physics, Jagiellonian University, Kraków, Poland Aberrant glycosylation, characteristic for cancer cells, can be partially reflected on the surface of ectosomes – small (100-1000 nm in diameter), membrane enclosed particles, that are released to the extracellular space by direct budding from the cell surface. This study investigated the surface glycosylation of cutaneous melanoma (CM) cells and corresponding ectosomes [1]. Primary (WM115, WM793) and metastatic (WM266-4, WM1205Lu) CM cells were cultured in vitro and ectosomes released by those four cells lines were isolated from conditioned media by sequential centrifugation. CM cells and CM-derived ectosomes were then stained with a panel of lectins (PHA-E, PHA-L, SNA, MAA, GNA, AAA), recognizing different glycoepitopes, and analyzed using flow cytometry. The presence of cancer-associated glycoepitopes was observed on the surface of CM ectosomes, however the percentage of lectin-positive ectosomes was smaller in comparison to the analogous lectin stainings of CM cells. It suggests that ectosomes derive from particular regions of cell membranes, therefore glycosylation of the entire cell surface is not identical with glycosylation of ectosome surface. For almost each staining (except GNA) the relative fluorescence intensity was higher for primary CM cell lines compared to ectosomes derived from isogenic cells lines of metastatic origin. Increased surface glycosylation of ectosomes derived from primary CM cells may be a way by which primary CM cells generate their invasive and metastatic potential. Finally the release of more heavily glycosylated ectosomes by primary CM cells may influence premetastatic niche formation through glycan- mediated binding of ectosomes with different ligands at the future sites of metastasis. ACKNOWLEDGEMENTS This work was supported by grants from the Polish National Science Centre (2013/11/B/NZ4/04315) and from the Jagiellonian University in Kraków (K/DSC/003978 and K/ZDS/007347). REFERENCES: 1. M. Surman, et al. Life Sciences vol. 207 p. 395-411, 2018.

36 O 9. Mechanisms and novel treatment approaches in allergen immunotherapy D. Van Elst1, M. Zemelka-Wiącek1, M. Jutel1

1Department of Clinical Immunology, Wroclaw Medical University, Wroclaw, Poland Asthma, allergic rhinits and food allergies are global health issues impressing a large social and economic burden on society. Our insight in immunoregulatory mechanisms has enabled the development of various treatment strategies for these allergic diseases. Allergen immunotherapy (AIT) was first reported 108 years ago by Leonard Noon and is still the only long-term curative treatment for patients suffering from allergic diseases [1]. Patients get exposed to increasing volumes of allergens, resulting in a drastic reduction of allergic symptoms upon exposure to the allergen. Effective AIT induces suppression of allergic inflammation, desensitization to the allergen and allergen-specific immune tolerance, followed by the production of allergen-specific regulatory T (Treg) and B (Breg) cells. Immune tolerance is elicited by targeting type II immune cells capable of producing IL-4, IL-5 and IL-13, cytokines which stimulate mast cell, basophil and eosinophil activation as well as IgE production [2, 3]. Despite the success of AIT, research into the treatment continues to improve efficacy, safety and convenience of the treatment [1, 4]. Recent developments include recombinant-allergen based therapy, intralymphatic or epicutaneous application of the allergen and a search for diagnostic biomarkers for personal medicine-type of treatment [5-7]. During my presentation I will review key mechanisms of AIT-triggered tolerance, advancements in standardization of AIT and novel treatment approaches. ACKNOWLEDGEMENTS The authors thank the supporting staff at Wroclaw Medical University with special thanks to their collegues at the department of clinical immunology, as well as their family and friends. REFERENCES: 1. M. Jutel, et al. Journal of Allergy and Clinical Immunology. vol. 136 nr. 3 p. 556-568, 2015. 2. M. Jutel, et al. Journal of Allergy and Clinical Immunology. vol. 137 nr. 2 p. 358-368, 2016. 3. A. Cezmi, et al. World Allergy Organization Journal. vol. 8 nr. 1 p. 1-12, 2015. 4. R. Valenta, et al. Journal of Allergy and Clinical Immunology. vol. 137 nr. 2 p. 351-357, 2016. 5. A. Frew. Clinical Immunology (Fifth Ed.). p. 1227-1235, 2019. 6. M. Jutel, et al. Allergy, Asthma and Immunology Research. vol. 8 nr. 3 p. 191-197, 2016. 7. M Berings, et al. Journal of Allergy and Clinical Immunology. vol. 140 nr. 5 p. 1250-1267, 2017.

37 3rd SESSION O 10. Organs-on-a-chip: an innovative tool in toxicological studies Ł. Lewandowski1, H. Milnerowicz1

1Department of Biomedical and Environmental Analyses, Faculty of Pharmacy with Division of Laboratory Medicine, Wrocław Medical University, Borowska 211, 50-556 Wrocław, Lower Silesian Voivodeship, Poland Pre-clinical in vivo studies on animal models are the main way to carry out experiments in toxicological studies. For years have they been utilized to study various aspects of development and homeiostasis, as a link between carrying out experiments on a Petri dish and developing clinical solutions for current challenges. However, the main drawbacks of animal in vivo studies are: the cost, high variability, and difficulty in interpreting the obtained results. Fortunately, a new tool for carrying out such studies has been invented. This technology, organs-on-a-chip (OoC) is aimed to simulate in vivo conditions, without using animals as a study model. OoC features microcompartments, which represent important tissues, organs, such as: liver, lung, brain, ovary, and physiological barriers between organs and blood vesicles (e.g. blood-brain barrier, blood-kidney barrier). The skeleton of each micro- compartment is made of highly biocompatible, durable materials. The core consists of adequate cell layers, originated from induced pluripotent stem cell line. The in vivo blood flow between organs and tissues, in case of OoC, has been substituted with pressure-controlled microfluid flow (laminar or turbulent, depending on the aim of the experiment) through synthetic microchannels. Thus – a stable supply of nutrients, hormones and cytokines, inter alia, between the cells, is maintained, resembling in vivo conditions. Different parameters can be assayed in nanodrops of microfluid, created with use of electromagnetic field. To sum up, OoC is a new technology, aimed to revolutionize preclinical in vivo studies. Not featuring animal models, it is highly cost-effective and somewhat easier to control. REFERENCES: 1. C. Moraes et al. Organs-on-a-Chip: A Focus on Compartmentalized Microdevices. Ann Biomed Eng 40, 1211-1227, 2012. 2. K. Ronaldson-Bouchard et al. Organs-on-a-Chip: A Fast Track for Engineered Human Tissues in Drug Development. Cell Stem Cell 22, 310-324, 2018. 3. D. Bovard et al. Organs-on-a-chip: A new paradigm for toxicological assessment and preclinical drug development. Toxicology Research and Application 1, 2397847317726351,2017.

38 O 11. How to predict the secondary from the primary structure of the protein? W. Szlasa

Faculty of Medicine, Wroclaw Medical University, Poland There are four levels of protein structure: primary, secondary, tertiary and quaternary. The last three highly depend on the amino acids sequence and the conditions of the system in which they are solvated, as well as other proteins – chaperones, which allow them to adopt the specific conformation. The structure can also vary in time, due to the fluctuations in the solution, for example the heat convection can locally change the environment of the polypeptide, resulting in change in the spatial arrangement of the amino acids chain [1]. Currently, to solve this problem, researchers use statistics methods as well as the ones which are based on homology in other proteins. The aim of this study was to invent a method which could be useful for the evaluation of the secondary structure of the protein based on the amino acids sequence. There were calculated specifically modified probabilities of the existence of each amino acid in each secondary structure. The obtained modifications, were based on the interactions between side chains of amino acids within alpha helices and beta strands. After dividing the chart into regions, I tried to estimate which structure could be found. There newly developed prediction method enabled to estimate the possible and potential endings of these proteins. The results presented in the study provide an excellent theoretical insight into the structure as well as better understanding of the proteins. They also reveal some interesting facts about the types of secondary structures. ACKNOWLEDGMENTS The work was created as part of the activity of the Student Research Group “Biology of Cancer Cell” at the Wroclaw Medical University (SKN No. K 148). REFERENCES: 1. Zhang Y. Protein structure prediction: when is it useful?. Curr Opin Struct Biol. 2009; 19(2): 145-55.

39 O 12. Calcium electroporation – a molecular dynamics simulation N. Szulc1, J. Wojciechowski2, M. Kotulska1, J. Kulbacka3, M. Tarek4

1Department of Biomedical Engineering, Wroclaw University of Science and Technology, Wrocław, Poland 2Department of Chemistry, Wroclaw University of Science and Technology, Wrocław, Poland 3Department of Molecular and Cellular Biology, Wroclaw Medical University, Wrocław, Poland 4CNRS Université de Lorraine, Nancy, France Electoroporation (EP) is a method using pulsed electric field (PEF), which creates short-living nanopores in the cell membrane [1]. It is widely used as gene transfection and drug delivery system in electrochemotherapy. Calcium electroporation is becoming more significant as an anti-cancer treatment. Furthermore, calcium is an important compound in biology, taking part in many processes such as: apoptosis or necrosis, signaling pathways etc [2]. The aim of our study was to simulate electroporation with calcium ions using two different sets of force field parameters. We compared classical approach with the one proposed by Ollila’s group [3]. This model takies into account polarizability effects by applying electronic polarization using mean field correction, known as the electronic continuum correction (ECC). This effects strongly contributes to electrostatic interactions in the system thus affect interactions between calcium ions and a membrane bilayer, which have influence on the transport across the membrane. We investigated interactions of calcium ions with phosphatidylcholine (POPC) bilayer in the presence of an external electric field as well as a calcium ions transport through electropores. We showed that ECC correction can influence distribution of calcium ions in the system as well as ion transfer across lipid membranes. REFERENCES: 1. J. C. Weaver, “Electroporation Theory: Concepts and Mechanisms” in Electroporation Protocols for Microorganisms, vol. 47, New Jersey: Humana Press, 1995, pp. 1-26. 2. T. G. Kinjo and P. P. M. Schnetkamp, “Ca2+ Chemistry, Storage and Transport in Biologic Systems: An Overview” 2013. 3. J. Melcr, H. Martinez-Seara, R. Nencini, J. Kolafa, P. Jungwirth, and O. H. S. Ollila, “Accurate Binding of Sodium and Calcium to a POPC Bilayer by Effective Inclusion of Electronic Polarization” J. Phys. Chem. B, vol. 122, no. 16, pp. 4546-4557, Apr. 2018.

40 O 13. Hypericin and phenothiazines – the use of computational methods to determine the structure and properties of drugs S. Szymański1, M. Polesiak1, I. Majerz1

1Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, Borowska 211A, 50-556 Wroclaw, Poland Computational methods allow approximation of geometry, physicochemical properties and electronic structure of the compounds with potential pharmaceutical applications. It is possible to plan strategies and reduce the cost of the experiment using these methods. Hypericin is naturally occurring antraquinone derivative found in Hypericium plants. Due to its broad pharmacological activity, hypericin has aroused a great interest in photodynamic therapy [1] and treatment of mild depression [2]. Hypericin can interact with many receptors thus, it is important to determine the spatial and electronic structure of this compound and to study its aromaticity. Phenothiazines belong to the group of neuroleptics which show affinity to many receptors, enzymes and proteins [3]. Phenothiazines are influenced by presence of the aminoalkyl chain attached to the nitrogen atom in the central ring [4]. It affect the geometry of the molecule by changing the angle between the planes of the side aromatic rings - the “butterfly” angle. Taking into account that the structure of organic molecules is a source of chemical and biological properties, we have undertaken a systematic theoretical study to analyze the structural parameters of phenothiazine and hypericin.

Fig. 1. Molecular structure of hypericin (left) and phenothiazine (right)

REFERENCES: 1. B. Chen et al. Cancer Lett. 2000, 150, 111-117. 2. V. Butterweck et al. H. Brain Res. 930, 21-29, 2002. 3. N.M. Richtand et al. Neuropsychopharmacology, 32(8): 1715-1726, 2007. 4. A.D. Mosnaim et al. American Journal of Therapeutics, 13(3): 261-273, 2006.

41 O 14. Pro-oxidative properties of cynamonic acid towards normal and cancer human cells D. Wiczew1, D. Przystupski2, J. Kulbacka3

1Wroclaw University of Science and Technology, Faculty of Chemistry, Wroclaw, Poland 2Faculty of Medicine, Wroclaw Medical University, Wroclaw, Poland 3Department of Molecular and Cellular Biology, Wroclaw Medical University, Wroclaw, Poland Many substances are considered antioxidants if they reduce cell's oxidation stress. Nevertheless, those substances which not directly deactivate reactive oxygen species, they have to work by a different mechanism; thus they can act differently depending on cell metabolism. In the study, we show that cynamonic acid, which lacks antioxidant properties in quantum chemical calculations, shows antioxidative capabilities in normal cell line CHO-K1 and prooxidative in the tumor cell line SKOV3.

42 4th SESSION O 15. Carotenoids as Source of Antioxidant Acitivity in Food Matrix L. Bogacz-Radomska1,2, U. Kaim1,2, R. Olędzki1,2, A. Orkusz1,2, J. Harasym1,2

1.Adaptive Food Systems Accelerator – Science Centre, Wrocław University of Economics, Poland 2. Department of Biotechnology and Food Analysis, Institute of Chemistry and Food Technology, Wrocław University of Economics, Poland e-mail address: [email protected] Carotenoids are organic tetraterpenoid pigments synthesized by plants and microorganism. These compounds exhibit bioactive and colour characteristic. Moreover carotenoids have antioxidant activity, that has a significant impact on human health. Carotenoids protect cells against oxidative stress. That results from modifications of their polyene backbone consisting of a series of conjugated C=C bonds, therefore the number of conjugated double bonds increase together with the addition of oxygen functional groups, in turn, alter the reactivity of carotenoids. Many studies have demonstrated a strong association between diets rich in fruit and vegetables and reductions in certain diseases, including some cancers and heart disease. Therefore they are applied in the food matrix. To the carotenoids applied in food belong carotenes, paprika extract, capsanthin and capsorubin, lycopene, lutein, annato, bixin and norbixin, beta-apo-8′-carotenal, astaxanthin. Carotenoid applications at the food matrix underline in regulation of the European Union. So far carotenoids are added (with some restriction) to: chees, fats, jam, jellies and marmalades and sweetened chestnut purée, processed potato products, breakfast cereals, fish and crustacean paste, precooked crustacean, smoked fish, edible ices, breath freshening microsweets, chewing gum, decorations, soups and broths, sauces, meat and fish analogues based on vegetable proteins, dietary foods, fruit wine and made wine, aromatised wine-product cocktails, other alcoholic drinks, ready-to-eat savouries and snacks, processed nuts, desserts. High dosage of carotenoids in food matrix act pro-oxidative and are adverse to human health. Therefore adherence of legal regulations has prime importance to food safety.

43 O 16. Synthesis and amidolytic activity of fragment of lunasin J. Kiszkiel1, P. Uścinowicz2, K. Wołosik3, A. Markowska2

1Students' Scientific Group of Department of Organic Chemistry,Medical University of Bialystok, Bialystok, Poland 2Department of Organic Chemistry, Medical University of Bialystok, Bialystok, Poland 3Laboratory of Cosmetology, Medical University of Bialystok, Bialystok, Poland Lunasin is a soybean-derived polypeptide which contains 43 amino acid residues. Lunasin is known for its pro-health properties, it has antihypertensive, anticholesterol, antioxidant and antitumour activities. Nevertheless, a key question is whether lunasin, a peptide with a relatively high molecular weight, is absorbed by the gastrointestinal system. A recent study showed that a number of peptides of hydrolysed lunasin can be found in human plasma after consumption of soy protein, among others HIMEK. For physiological processes of tissue remodelling, the proteolytic degradation process is so important that in the critical mechanism of tumour invasion can contribute to the inhibition of its growth and metastasis. The plasminogen activation system (PA) plays an important role in degradation reactions. It was found that the level of expression of the PA enzyme system is associated with poor or good prognosis of breast, kidney, colon, stomach and brain cancers. The aim of this work was to synthesize HIMEK peptide and its amide analogs with N-terminal of palmitoyl, acetyl and lipoic fragments and to evaluate the inhibition of proteolytic enzymes of the plasminogen activator system (PA). The peptides were synthesized manually on solid phase synthesis using the standard Fmoc- based strategy. Effects of peptides on the activity of PA system were investigated using a chromogenic synthetic substrate in amidolytic test. Based on our research, the results and conclusions of our work will be discussed.

44 O 17. Changes In Lysozyme’s II-Structure As A Result Of Its Interaction With A Gold Surface – Preferred Conditions for Lysozyme’s Aggregates Formation in Neurodegenerative Disease Development P. Komorek1, I. Brand2, B. Jachimska1

1Jerzy Haber Institute of Catalysis and Surface Chemistry Polish Academy of Sciences, Krakow, Poland 2Department of Chemistry, Carl von Ossietzky University of Oldenburg, Oldenburg, Germany One of the hypothesis combines the development of the neurodegenerative disease with changes in protein II-structure and its aggregation. As lysozyme is a protein associated with Alzheimer disease, therefore it is anticipated that it has the potential to be used as a new biomarker at therapeutic target for Alzheimer disease in the near future. In this work, the adsorption of lysozyme onto the gold surface under different conditions (pH, ionic strength and concentration) was investigated using the optical method – Multi-Parameter Surface Plasmon Resonance (MP-SPR) and mechanical method – Quartz Crystal Microbalance (QCM-D). Experimental data shows that maximum adsorption efficiency was obtained at the pH=10, which corresponds to lysozyme’s isoelectric point. Comparison of results obtained by MP-SPR and QCM-D allows calculating the hydration level of lysozyme’s layer. The orientation of lysozyme’s molecules onto the gold surface was estimated according to Random Sequential Adsorption (RSA) model. The stability of lysozyme’s conformation was examined using Polarization-Modulation Infrared Reflection Absorption-Spectroscopy (PM-IRRAS) method. Analysis of the PM-IRRAS data shows that the interactions between lysozyme and the gold surface caused changes in its II-structure in comparison with conformation in the solution obtained from Circular Dichroism experiments. Additionally, the II-structure of lysozyme under different electric potential conditions was investigated. The presence of electric potential results in strong misfolding of lysozyme in low pH conditions (pH=4). This great changes in lysozyme’s II-structure are linked to the formation of aggregates. ACKNOWLEDGEMENTS This work was partially supported by project NCN OPUS 2016/23/B/ST5/02788, InterDokMed POWR.03.02.00-00-I013/16, and NAWA PPN/BIL/2018/1/00103 REFERENCES: 1. B. Jachimska, A. Kozlowska, A. Pajor-Swierzy, “Protonation of Lysozymes and Its Consequences for the Adsorption onto a Mica Surface”, Langmuir, 28, 2012, 11502-11510. 2. K. Kubiak–Ossowska, M. Ćwięka, A. Kaczyńska, B. Jachimska, P. Mulheran, “Lysozyme adsorption at a silica surface using simulation and experiment: effects of pH on protein layer structure.”, Physical Chemistry Chemical Physics (PCCP), 2015, 17, 37, 24070-77.

45 O 18. Anti-cancer effect of photodynamic therapy and electroporation mediated by curcumin on the A375 melanoma cell line S. Kwiatkowski1, W. Szlasa1, V. Novickij2, D. Przystupski1, K. Kotowski1, A. Kiełbik1, P. Wawryka1, J. Saczko3, J. Kulbacka3 1Faculty of Medicine, Wroclaw Medical University, Wroclaw, Poland 2Institute of High Magnetic Fields, Vilnius Gediminas Technical University, Vilnius, Lithuania 3Department of Molecular and Cellular Biology, Wroclaw Medical University, Wroclaw, Poland Malignant melanoma is a cancer arising from neoplastically transformed melanocytes. New medicines are being sought to effectively fight against melanoma. The development of therapies and therapeutics that would bring a satisfactory therapeutic and low adverse effects is necessary. The promising natural compound presenting proapoptotic and anti-cancer properties is curcumin. Curcumin can act also as a photosensitizer in photodynamic therapy (PDT) [1] and is suitable agent for electroporation – method enhancing cell permeability for cancer treatment [2]. In the present study, curcumin was investigated in PDT using human skin malignant melanoma cell line (A375) and normal human fibroblasts from primary culture as a control cells. The cells were maintained in culture and then treated with curcumin at concentrations of 5-50 μM for 24 and 48 hours. After incubation, the cells were irradiated with blue light (20 J/cm2) for 5 minutes. Simultaneously, the melanoma A375 cells were treated with pulsed electric field (PEF) alone and in combination with curcumin. The following electroporation (EP) parameters were applied: 200 and 1200 mV/cm, 100 μs by 8 pulses for every case. The efficacy of photodynamic effect and electroporation mediated by curcumin was evaluated by the viability assay (MTT). The obtained results showed that PDT with curcumin can increase a number of apoptotic and necrotic cells in comparison to incubation with curcumin without irradiation. Our study demonstrated that PEF provoked additive reaction with curcumin and inhibited the proliferation of A375 cells. The proposed protocol seems to be promising in human melanoma treatment. ACKNOWLEDGEMENTS The research was financed partially by the Student Scientific Group “Biology of cancer cells” (Wroclaw Medical University) and by “Najlepsi z Najlepszych 3.0!” program of Polish Ministry of Science and Higher Education and Statutory Funds of Department of Molecular and Cellular Biology. REFERENCES: 1. H. Abrahamse, et al. Biochem J. vol. 473(4) p. 347-364, 2016. 2. Ch. Lu, et al. Onco Targets Ther. vol. 11 p. 4723-4732, 2018.

46 O 19. Oximes derived from natural volatile compounds as antimicrobial active agents A. Surowiak1, S. Lochyński1,2, D. Strub1

1 Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology, Wyb. Wyspiańskiego 27, 50-370 Wrocław, Poland 2Institute of Cosmetology, Wroclaw College of Physiotherapy, ul. Kościuszki 4, 50-038 Wrocław, Poland Bacterial and fungal pathogenes are significant threat to human well-being, new active agents against microorganisms are currently being sought to overcome the challenge of antimicrobial resistance. We wanted to address this societal challenge by evaluating the antimicrobial activity of low-molecular oximes, which haven’t been extensively studied with regard to this issue. Fifty-three oximes and their substrates were screened in vitro for their growth inhibitory activity against seven strains of microorganisms: E. coli (ATCC 10536), S. aureus (ATCC 6538), E. hirae (ATCC 10541), P. aeruginosa (ATCC 15442), L. pneumophila (ATCC 33152), A. brasiliensis (ATCC 16404), and C. albicans (ATCC 10231). The first step of our research was testing the growth inhibition of microorganisms using the paper disc diffusion method. Three antibiotics (netilmicin, fluconazole, and ofloxacin) were used as reference controls for the tested microorganisms. The second step of our research was determining the minimal inhibitory concentration (MIC) for substances that in the first step showed medium or strong activity against certain strains. The MIC parameters were determined using the micro-plate Alamar Blue® assay. The trans-cinnamaldehyde and (±)-citronellal, propiophenone, α-isomethylionone, pseudoionone, α-hexylcinnamaldehyde, (-)-fenchone and (±)-campher oximes showed inhibitory activity against moulds and yeasts, whereas only m-tolualdehyde, (±)-citronellal and dihydro-α-ionone showed inhibitory activity against Gram-negative bacteria. The o-Tolualdehyde, p-tolualdehyde, α-hexylcinnamaldehyde, propiophnone, α-isomethylionone, pseudoionone, benzaldehyde, geranyl acetone, (+)-pulegone, dihydrojasmone, m-anisaldehyde, (-)-fenchone, and (+)-fenchone oximes showed strong inhibitory activity against Gram-positive bacteria. The most significant MIC parameter (23,44 µg/ml) was that of α-isomethylionone oxime against E.hirae. ACKNOWLEDGEMENTS This work was supported by the project “Synthesis of new fragrances from raw materials of a natural origin with application in perfumery, cosmetics and household chemistry” (SYNFRA); grant no. LIDER/4/0099/L-7/15/NCBR/2016; which is financed by the National Centre for Research and Development within the LIDER Programme.

47 5th SESSION O 20. Impact of demographic factors on prevalence, severity level and course of urticaria in patients in Department of Allergy, Clinical Immunology and Internal Medicine in 2012-2018 years Z. Bartuzi1, J. Durślewicz,2 J. Karczewska1, N. Ukleja-Sokołowska1, R. Zacniewski1

1Department of Allergy, Clinical Immunology and Internal Medicine, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun, Poland 2Department of Clinical Pathomorphology, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun, Poland Urticaria is a common skin disorder. Pathogenesis is multifactorial. Pathognomonic wheals, pruritus and angioedema are triggered by mast cell-driven vascular reaction in response to external allergic, chemical and physical factors. The aim of this study was urticaria develop risk factors evaluation and contribution this factors in exacerbation of the disease. Retrospective study based on medical records of patients of Department of Allergy, Clinical Immunology and Internal Medicine, Nicolaus Copernicus University in Torun, Poland in 2012-2018. We analyzed medical records of 174 patients (127 female, 47 male) in terms of demographic conditions, details of hospitalizations, accompanying disorders and allergies. 85 (49%) patients (15 male, 70 female) demonstrated allergic sensitization to minimum one allergen. In female group the most common food allergen was rye flour and inhalant allergen was dust mite. In group of male patients it was celery and house dust mite. Twice as many patients live in a city agglomeration than in a countryside, with predominance of sensitization in males in the countryside (50%) rather than in city’s (33%). Analysis didn’t show statistically significant correlation between urticaria and accompanying disorders. It is a known fact the clinical picture of urticaria is influenced by food allergens, pollens and air pollution. Furthermore, there’s lots of other unknown factors, which influence on incidence the exacerbation level of urticaria. Significantly higher frequency of urticaria in population of women could suggest that using lots of cosmetics, perfume and household chemicals is one of the factors of higher prevalence of urticaria in females.

48 O 21. An association between MMP-2, MMP-9, TIMP-4 and stable coronary artery disease, hypertension and diabetes M. Banaszkiewicz1, W. Kuliczkowski2, I. Bil-Lula1

1Division of Clinical Chemistry, Department of Medical Laboratory Diagnostics, Wroclaw Medical University, Wroclaw, Poland 2Department and Clinic of Cardiology, Wroclaw Medical University, Wroclaw, Poland Coronary artery disease (CAD) is accompanied by many diseases, including hypertension (HT) and diabetes (DM). Metalloproteinases 2, 9 (MMP-2, MMP-9) and tissue inhibitor of MMPs (TIMP-4) are involved in pathophysiology of these diseases. The aim was to study an influence of CAD, HT and DM on plasma MMP-2, MMP-9 and TIMP-4 levels. The study involved 70 CAD patients admitted for coronary angiography and 15 healthy subjects. 32 CAD patients suffered from both HT and DM, 25 had HT only, 2 had DM only, and 11 had CAD without HT and DM. Whole blood samples were collected prior to angiography. MMP-2, MMP-9 and TIMP-4 levels were estimated by ELISA. CAD patients showed significant increased level of TIMP-4 and decreased level of MMP-2 in comparison to healthy control (p = 0.011, p = 0.037, respectively). Internal group comparison showed that CAD DM+ patients presented higher MMP-2 level than DM- patients (p<0.001). MMP-2, MMP-9 and TIMP-4 concentrations were not different in HT+ or HT- groups. Multiple regression analysis of influence of independent variables such as CAD stage, DM and HT on MMP-2, MMP-9 and TIMP-4 showed that only DM was independently associated with higher level of MMP-2 (β = 0.42 , R2 = 0.17, p<0.001). Data showed that patients with CAD have higher TIMP-4 and lower MMP-2 concentration regardless of HT and DM. DM occurrence is independently associated with higher MMP-2 concentration in those patients. HT had no effect on MMP-2, MMP-9 and TIMP-4 levels. FOUNDATION: National Science Centre no:2014/15/13/NZ3/04865

49 O 22. Graft-versus-host disease in posttransplantational pediatric patients G. Bis1, K. Sondaj1, A. Żarczyńska1, I. Zendran1, W. Szlasa1, E. Barg2

1Faculty of Medicine, Wroclaw Medical University, Wroclaw, Poland 2Faculty of Pharmacy with Division of Laboratory Diagnostics, Department of Basic Medical Sciences, Wroclaw Medical University, Wroclaw, Poland Graft-versus-host disease (GvHD) is a medical complication following the receipt of transplanted tissue from a genetically different person. GvHD is characterized by the damage to the skin, gastrointestinal tract’s mucosa, liver and other organs. In order for GvHD to occur transplant recipient must be immune compromised and histoincompatible with immune- competent graft. The problem does not only concerns to adults, but also to pediatrics patients[1]. The aim of the project was to investigate possible correlations between the basic factors of human body (mass, height, BMI, age and sex) with the posttransplantional state of the patient. Archivised in “Przylądek Nadziei” data regarding 151 patients subjected to stem cells transplantation were gathered and statistically analyzed. Among them 71 patients were classified in the first two classes of acute GvHD (aGvHD). Characteristics of each group of GvHD has been determined e.g. in the second class of aGvHD the vast majority (28/33) of patients expressed third class of skin disorders. Also it has been noticed that in low classes of acute GvHD, there were no symptoms of liver involvement. Investigations revealed that children above 7 years old who had graft were more likely to develop endocrine disorders (p<0,02) as well as it was more probable to find anti-CMV antibodies in their blood (p<0,04). Performed investigation included reference to scientific papers and consultations with medical professionals. Obtained results could be useful for physicians who deal everyday with pediatric patients, qualified for transplantation. ACKNOWLEDGMENTS The work was created as part of the activity of the Student Research Group “Basic Medical Sciences” at the Wroclaw Medical University. REFERENCES: 1. Rocha V, Wagner JE, Sobocinski KA, et al. “Graft-Versus-Host Disease in Children Who Have Received a Cord-Blood or Bone Marrow Transplant from an HLA-Identical Sibling” N Engl J Med. 2000;342(25):1846-1854. doi:10.1056/NEJM200006223422501.

50 O 23. Studies of haemostatic dressings reaction on fibroblasts and microorganisms K. Kołodziejczyk1, M. Szymonowicz1, J. Nowicka², M. Pajączkowska², A. Rusak³, B. Żywicka1, M. Dobrzyński⁴, Z. Rybak1 1Department of Experimental Surgery and Biomaterial Research, Wroclaw Medical University, Wroclaw, Poland 2Department of Microbiology, Wroclaw Medical University, Wroclaw, Poland 3Department of Histology and Embryology, Wroclaw Medical University, Wroclaw, Poland 4Department of Conservative Dentistry and Pedodontics, Wroclaw Medical University, Wroclaw, Poland Products with direct hemostatic effects on gingiva, that contain various chemical compounds, are appropriate to rapidly stop the bleeding in the oral cavity. The vasoconstrictor preparations may contain astringents. The degree of biocompatibility and the direct impact on the cells are associated with the form as well as the chemical composition of the product. The aim of the research was to determine whether and how aluminium chloride based materials affect the cell line as well as bacterial strains and fungi. Following materials were selected in the study: Alustin liquid (20% AlCl3 * 6H2O, chinosol), Hemostat gel (20% AlCl3 * 6H2O, chinosol) Racestyptine liquid (25% AlCl3 * 6H2O, oxychinol) and Traxodent gel. The in vitro cytotoxic activity of preparations extracts was performed on murine L929 fibroblasts. The degree of toxicity was determined by assessing cell survival by colorimetric determination of their metabolic activity using the SRB test and cell morphology changes using the 0-4 scale. In vitro microbiological examinations were performed on selected oral microorganisms: Candida albicans (ATCC 90028), Streptococcus mutans (ATCC 25175), Lactobacillus rhamnosus (ATCC 9595). The number of colony forming units (Cfu / ml) and survival of microorganisms were determined. The smallest concentration of the preparation (MIC) that inhibited microbial growth was determined. Alustin, Hemostat. Racestyptine Traxodent do not show cytotoxic activity towards murine L929 fibroblast cells. However, they result in decreased cell survival and intracellular granularity as well as changes in the cell nucleus not exceeding 2 degrees of toxicity. All applied hemostatic preparations inhibited S. mutans growth. Alustat and Hemostat showed a complete cfu/ml reduction of all analyzed strains despite high MIC values. The smallest efficiency against the tested strains had Recystypine and Traxodent. ACKNOWLEDGEMENTS: The work was performed within the scientific activity ST.B08.17.025 of the Wroclaw Medical University.

REFERENCES: 1. ISO 10993-5 2009, Biological evaluation of medical devices. Part 5: Tests for in vitro cytotoxicity. 2. Skehan, Philip, et al. New colorimetric cytotoxicity assay for anticancer-drug screening. JNCI: Journal of the National Cancer Institute 82.13 (1990): 1107-1112. 3. Mounyr Balouirin , Moulay Sadiki, Saad Koraichi Ibnsouda. Methods for in vitro evaluating antimicrobial activity: A review. Journal of Pharmaceutical Analysis 6 (2016) 71-79. 4. G. Morea, T.E. Tshikalangea, N. Lall, F. Botha, J.J.M. Meyer. Antimicrobial activity of medicinal plants against oral microorganisms. Journal of Ethnopharmacology 119 (2008) 473-477. 5. Elshikh, M., Ahmed, S., Funston, S. et al.Resazurin-based 96-well plate microdilution method for the determination of minimum inhibitory concentration of biosurfactants Biotechnol Lett (2016). 6. Szymonowicz M, Korczyński M, Dobrzyński M, Zawisza K, Mikulewicz M, Karuga-Kuźniewska E, Żywicka B, Rybak Z, Wiglusz RJ. Cytotoxicity evaluation of high-temperature annealed nanohydroxyapatite in contact with fibroblast cells. Materials 2017;10(6):13. 7. Kirstein K, Kołodziejczyk K, Cykowska M, Dobrzyński M, Szymonowicz M, Skalec A, Szklarz M, Chrószcz A, Janeczek M, Rybak Z. Bioresorbable materials in dental surgery. Pol J Environ Stud 2016;25(6A):87-90.

51 O 24. The use of thermography in experimentally induced pleurisy induced by low dose of TCDD P. Kuropka1, M. Dobrzyński2, K. Dudek3, M.Tarnowska4, A. Leśków4, R. J. Wiglusz5

1Department of Histology and Embriology, Wroclaw University of Environmental and Life Sciences, Norwida 31, 50-375 Wroclaw, Poland 2Department of Conservative Dentistry and Pedodontics, Wroclaw Medical University, Krakowska 26, 50-425 Wroclaw, Poland 3Faculty of Mechanical Engineering, Technical University of Wroclaw, Lukasiewicza 5, 50-371 Wroclaw, Poland 4Department of Nervous System Diseases, Wroclaw Medical University, Bartla 5, 51-618 Wroclaw, Poland 5Institute of Low Temperature and Structure Research, Polish Academy of Sciences, Okolna 2, 50-422 Wroclaw, Poland Dioxins are a group of compounds with strong toxic properties, present among others in the air. Its influence on tissues is widely described but there is little information available in the literature on its effects in the respiratory system. Thermography is a non-invasive and quite precise method of measuring thermal radiation emitted by physical bodies in the range of temperatures encountered in natural conditions. Therefore the aim of this study was to compare the results obtained from themography with the histological picture of rat lungs after administration of TCDD. 8 female from Buffalo inbreeding strain were used. All rats were kept in the same conditions. The animals from experimental groups received 0,5 µg/kg b.w. of TCDD into the areas of right intercostal space. 24 and 48 hours after injection, rats were chemically depilated with a cream and 2h later the measurement of temperature was performed. Then the rats were euthanatized and lung samples were taken for histological analysis. In the experimental group, in which the pleurisy was developed both the average maximum and the average minimum temperature were the highest after 48 hours after injection. Several proliferative changes were detected in the lungs, including alveolar epithelial hyperplasia, hyperaemia and oedema. Intense migration of macrophages and lymphocytes was observed followed by increased proliferation of epithelial cells. These changes were the most intensive 48h after injection. The study confirms that the developing inflammation process can be studied at the early stages by this visual non-invasive method. ACKNOWLEDGEMENTS The authors would like to thank Prof. Ireneusz Całkosiński (1951–2017), PhD, for his inspiration, helpful advice and assistance in assuring the welfare of animals. REFERENCES: 1. M. Dobrzyński, et al. “Co-expression of the aryl hydrocarbon receptor and estrogen receptor in the developing teeth of rat offspring after rat mothers' exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin and the protective action of α-tocopherol and acetylsalicylic acid” Adv Clin Exp Med. doi: 10.17219/acem/99613, 2019 Jan 24. 2. MM Leijs et al. “Exposure to Environmental Contaminants and Lung Function in Adolescents-Is There a Link?” Int J Environ Res Public Health vol. 15(7), pii: E1352. doi: 10.3390/ijerph15071352, 2018 Jun 27. 3. M Sarill et al. “The aryl hydrocarbon receptor suppresses cigarette-smoke-induced oxidative stress in association with dioxin response element (DRE)-independent regulation of sulfiredoxin 1” Free Radic Biol Med. vol. 89, pp. 342-357, 2015 Dec.

52

POSTER PRESENTATIONS

P 1. Epigenetic modifications in adipose tissue associated with obesity A. Alama1, D. Pawełka2, A. Myszczyszyn3, M. Małodobra-Mazur1

1 Molecular Techniques Unit, Wrocław Medical University, Wrocław, Poland 2 First Department and Clinic of General, Gastroenterological and Endocrinological Surgery, Wrocław Medical University, Wrocław, Poland 3 First Department and Clinic of Gynaecology and Obstetrics, Wrocław Medical University, Wrocław, Poland Obesity is a serious and constantly developed problem of modern civilization. This problem affects both adults and children. Moreover, obesity is increasingly associated with serious metabolic disorders such as insulin resistance or type 2 diabetes (T2D). Numerous studies implicate obesity with epigenetic modification within DNA, especially with methylation. In addition, many scientific reports indicate a functional relationship between increased methylation of DNA and the expression of genes encoding proteins involved in important metabolic pathways, which could explain the relationship between obesity and metabolic diseases such as insulin resistance1,2. In present study, we analyzed the influence of obesity on the global DNA methylation and expression of genes encoding epigenetic modifying enzymes in human adipose tissue. Both types of adipose tissue, visceral (VAT) and subcutaneous (SAT), were subjected to the examination, which allowed to assess the metabolic differences between both types. We observed that the global methylation within DNA was increased in obese individuals, both in visceral and subcutaneous adipose tissue. In addition, increased methylation was accompanied by an increase in the expression of methyltransferases (DNMT), which would confirm the participation of these proteins in the generation of methylation within DNA. The results also showed that in patients with insulin resistance DNA methylation was higher than in insulin sensitive patients, which could indicate that the relationship between obesity and insulin resistance may result from epigenetic modifications. REFERENCES: 1. Drogan D, Boeing H, Janke J, et al. “Regional distribution of body fat in relation to DNA methylation within the LPL, ADIPOQ and PPARγ promoters in subcutaneous adipose tissue” Nutr Diabetes. 2015; 5:168. 2. Zheng LD, Linarelli LE, Liu L et al. “Insulin resistance is associated with epigenetic and genetic regulation of mitochondrial DNA in obese humans” Clin Epigenetics. 2015; 7:60.

55 P 2. Capillary electrophoretic separation of human albumin into subfractions A. Biedroń1, M. Kielar2, P. Dumnicka3

1Scientific Students’ Group of Laboratory Diagnosticians at the Department of Medical Diagnostics, Faculty of Pharmacy, Jagiellonian University Medical College, Kraków, Poland 2Medical Diagnostic Laboratory with a Bacteriology Laboratory, St. Louis Regional Children's Hospital, Kraków, Poland 3Department of Medical Diagnostics, Faculty of Pharmacy, Jagiellonian University Medical College, Kraków, Poland In various disease states, albumin undergoes posttranslational modifications, e.g. glycation in diabetes, or carbamoylation in end-stage renal disease. Moreover, various compounds characterized by poor water solubility bind non-covalently to serum albumin. Some of the modifications may be regarded as the prognostic/severity markers of disease. The modifications may alter electrophoretic characteristics of albumin. The aim of the study was to assess the capability of capillary electrophoresis to separate albumin subfractions resulting from above-mentioned modifications of its structure. Capillary electrophoresis system Prince 450 (Prince Technologies, Emmen, The Netherlands) equipped with UV-VIS detector and DAx software (Van Mierlo Software Consultancy, Eindhoven, The Netherlands) was used. Electrophoresis was carried out in bare silica capillary 60 cm long, using voltage of 20-30 kV. Protein fractions were detected at 214 nm. We used several alkaline (pH 9-10) buffers containing N-methyl-D-glucamine (concentrations 10-100 mmol/l) and carboxyl acids (caprylic, lauric, palmitic) in various concentrations (5-60 mmol/l). Human serum diluted 10-times in water was a sample. The ability of various buffers to separate albumin into subfractions was tested. The use of low concentrations of N-methyl-D-glucamine and low concentrations of short-chain caprylic acid resulted in a separation of human serum into five or six fractions (albumin, alpha-1 and 2, beta-1 and 2, gammaglobulins), however, albumin presented as a single fraction. The addition of longer-chain fatty acids (lauric, palmitic) resulted in a much wider peak of albumin, separated into two to four subfractions. Further studies are planned to evaluate the identity and clinical significance of albumin subfractions.

56 P 3. Interaction of neobavaisoflavone with lipid bilayers – microcalorimetric study M. Błaszczyk1, O. Wesołowska1

1 Department of Biophysics, Wrocław Medical University, Chałubińskiego 10, 50-368 Wrocław, Poland Neobavaisoflavone (NBIF) is a phenolic compound of natural origin, isolated from seeds of medicinal plant Psoralea corylifolia [1]. This herbal medicine has been traditionally used for treating various skin diseases as vitiligo, alopecia areata, leukoderma, and psoriasis [2]. NBIF is known for many biological activities, including anticancer, anti-inflammatory, antioxidant [1], and neuroprotective activity [2]. Differential scanning calorimetry (DSC) allows to investigate the influence of various compounds on the thermal properties of phospholipid bilayers. The aim of the study was to investigate the effect of NBIF on model phospholipids bilayers formed from phosphatidylcholine. Two types of phosphatidylcholine were used, DMPC and DPPC, differing by the length of their hydrocarbon chains. Neobavaisoflavone affected thermal properties of phosphatidylcholine bilayers in concentration-dependent manner. Pretransition was not recorded in samples containing NBIF. The obtained thermograms showed that main phospholipid transition in the presence of NBIF occurred at a lower temperature compared to pure phospholipid. Transition peaks of samples containing NBIF became asymmetric and were widened relatively to the increase in their half-height width. Also, the enthalpy change of main phospholipid phase transition was affected by NBIF. According to our best knowledge, this is the first study on interaction of NBIF with lipid membranes. Interaction with cell membranes and modification of their biophysical properties like fluidity or permeability are likely to contribute to the biological activity of xenobiotics, such as NBIF. REFERENCES: 1. MA Tao et al. Chin J Nat Med, 15(4) p.281-287., 2017 2. Y.J. Kim, H. Lim, J.Lee.Molecules, 21p. 1076, 2016.

57 P 4. Stability of some polyphenols in vitro gastrointestinal digestion A. Bodalska1, A. Kowalczyk1, I. Fecka1

1Department of Pharmacognosy and Herbal Medicines, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, Poland Phenolic compounds are natural secondary metabolites commonly occurred in plant derived foods like fruits, vegetables and juice as well as in medicinal plants. Polyphenols are the subject of increasing scientific interest because of their possible beneficial effects on human health. It has been proved that those compounds possess many bioactive properties, like against oxidative damage, antiphlogistic, antineoplastic activity [1, 2]. The aim of the study was to review findings in polyphenols’ stability after in vitro digestion. Scientist have conducted the research into polyphenols stability in commercially available products [3,4]. Fruit and vegetable juices (e.g. carrot, beetroot, tomato, orange, apple) were examined. Juice samples were mixed with pepsin, NaCl and HCl solution and incubated in 37 degrees for one hour in to mimic the process of gastric digestion. Second phase – intestinal digestion – included incubation in 0.9 M NaHCO3 solution with bile salts and pancreatin – lipase solution for two hours [5]. Folin-Ciocalteu method was used to measure total polyphenolic content. Both gallic and ferulic acids were used as a polyphenol equivalent to evaluate the difference between original and after in vitro digestion juice. Studies have shown that the level of total polyphenols have significantly increased in most samples after gastric digestion. Pancreatic in vitro digestion led to statistically significant decrease of polyphenols seven of the 28 analysed juices. Despite this, the overall antioxidant activity of most samples remained relatively high. REFERENCES: 1. Martins Natalia et al. “In vivo antioxidant activity of phenolic compounds: Facts and gaps”. Trends in Food Science & Technology 48, p. 1-12, 2016. 2. Roleira Fernanda et al. “Plant derived and dietary phenolic antioxidants: Anticancer properties” Food Chemistry, 183, p. 235-258, 2015. 3. Wei Quan et al. “Stability of the phenolic compounds and antioxidant capacity of five fruit (apple, orange, grape, pomelo and kiwi) juices during in vitro – simulated gastrointestinal digestion” International Journal of Food Science and Technology 53, p. 1131–1139, 2018. 4. Peter C. Wootton-Beard et al. “Stability of the total antioxidant capacity and total polyphenol content of 23 commercially available vegetable juices before and after in vitro digestion measured by FRAP, DPPH, ABTS and Folin–Ciocalteu method” Food Research International 44, p. 217-224, 2011. 5. Aura Anna-Marja “In vitro digestion models for dietary phenolic compounds” VTT Publications 575, 2005.

58 P 5. Pro-vitamin A of Microbiological Origin L. Bogacz-Radomska1,2, U. Kaim1,2, A. Orkusz1,2, R. Olędzki1,2, J. Harasym1,2

1Adaptive Food Systems Accelerator – Science Centre, Wrocław University of Economics, Poland; 2 Department of Biotechnology and Food Analysis, Institute of Chemistry and Food Technology, Wrocław University of Economics, Poland; e-mail address: [email protected] Carotenoids, whose structure is identical to the retinol molecule, present vitamin A activity and among this group, β-carotene reveals the highest bioactivity as vitamin A precursor. However, β-carotene conversion to vitamin A is incomplete and this compound shows only 1/6 of retinol activity, which means that 1 mg of retinol is equivalent to 6 mg of β-carotene. Among all the known methods of obtaining β-carotene, the microbial biosynthesis is of particular interest. The need for commercial production of natural pigment, including β-carotene, has led to intensive research in this area. Much attention is drawn to Dunaliella spp. algae that have the capacity to produce large amounts of β-carotene as well as alga Eustigmatos cf. Polyphem. The ability of carotenogenesis was also found in some bacteria Rhodococcus maris and Rhodobacter sphaeroides. Many works are focused on the different species of yeast synthesizing β-carotene, due to their high growth rate. On the industrial scale, β-carotene is obtained in molds Blakeslea trispora and alga Dunaliella salina cultivations. β-Carotene biosynthesis by Blakeslea trispora uses the opposite sex “+” and “−”. The cultivation of alga Dunalella salina needs special conditions like intense sunlight and relatively saline water. Cultures are conducted in three main stages: inoculum multiplication, biomass biosynthesis, and carotenoids extraction. Depending on the final form of β-carotene, biomass is subjected to appropriate technological operations. β-Carotene has numerous biological functions in the human body and because human is not able to synthesize any of them, it is necessary to supply these valuable compounds with food or pharmaceuticals

59 P 6. Anti-glioma activities of garlic compounds – an in vitro study A. Choromanska1, J. Saczko2, J. Kulbacka2, J. Rossowska3, P. Surowiak4

1Department of Medical Biochemistry, Wroclaw Medical University , Chalubinskiego 10 ,50-368, Wroclaw, Poland 2Department of Molecular and Cellular Biology , Wroclaw Medical University , Borowska 211A ,50-556, Wroclaw, Poland 3Institute of Immunology and Experimental Therapy Polish Academy of Sciences, Weigla 112, 53-114, Wroclaw, Poland 4Department of Human Morphology and Embryology, Wroclaw Medical University , Chalubinskiego 6a 50- 368, Wroclaw, Poland Gliomas are tumors of the central nervous system derived from glial cells. Despite the continuous development of medicine gliomas are tumors with a very poor prognosis and low curability. The aim of our study was to evaluate the anti-glioma activity of oil-soluble compound derived from garlic - diallyl disulfide and garlic oil. Tests were performed on four glioma cell lines with different levels of progression. We determined the IC50 for both substances and evaluated apoptosis inductions using flow cytometry. We observed that garlic oil was toxic to all tested cell lines in low doses. At the same time it effectively induces apoptosis. Diallyl disulfide induced cytotoxicity in two of the four tested lines and induced apoptosis only in the anaplastic glioma cells. Garlic oil has a more potent anti- glioma cells activity. The results obtained are promising and they will be the basis for further study. ACKNOWLEDGEMENTS This work was supported by the Grant for Young Scientist Wroclaw Medical University, no. STM.A040.18.013

60 P 7. Game animals as a source of Serratia sp. - an opportunistic pathogen for humans

G. Cieniuch1, A. Korzeniowska-Kowal2, A. Wzorek2, K. Morka1, J. Bania3, J. Bystroń3, G. Bugla-Płoskońska1 1Department of Microbiology, Institute of Genetics and Microbiology, Faculty of Biological Sciences, University of Wroclaw, Wroclaw, Poland 2Polish Collection of Microorganisms, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland 3Department of Food Hygiene and Consumer Health Protection, Wrocław University of Environmental and Life Sciences, Wroclaw, Poland Genus Serratia sp. contains species that are potentially pathogenic to humans [1]. Bacterial examination of game animals meat is important, due to its pathogenic potential for humans [2]. The aim of the study was the determination of Serratia sp. occurrence within game animals tonsils, i.e. wild boars (Sus scrofa) and roe deer (Capreolus capreolus). Swabs from game animals tonsils were obtained from Wielkopolska Province, Poland. A total 698 of tonsils swabs were obtained from wild boar and roe deer. As a result of use of medium CIN (Biocorp) from 698 wild boars and roe deer tonsils swabs we have obtained 139 of Serratia sp. isolates. MALDI-TOF MS allowed to identify the following species: S. liquefaciens, S. fonticola, S. marcescens and S. plymuthica. The most common identified species within genus Serratia sp. was S. liquefaciens (n=100/139). The other species were identified with the following frequency: S. fonticola (n=31/139), S. marcescens (n=5/139) and S. plymuthica (n=3/139). Our study demonstrated that wild boars and roe deer could be a reservoir of an opportunistic pathogen strains of Serratia sp. for humans. Serratia sp. despite the fact that it is opportunistic pathogens bacteria for humans, it is also isolated from various places in the environment. Strains of S. fonticola and S. plymuthica were isolated from water [3,4]. S. liquefaciens is also an etiological factor of mastitis in dairy cows [5], while S. marcescens are pathogen for insect too [6]. In the literature there is no information about the isolation of S. plymuthica, S. fonticola, S. liquefaciens and S. marcescens from wild boars and roe deer tonsils. REFERENCES: 1. S. D. Mahlen, et al. Clinical Microbiology Reviews vol. 24 p. 755-791, 2011. 2. X. J. Meng, et al. Philosophica Transactions of The Royal Society B vol. 364 p. 2697-2707, 2009. 3. F. Gavini, et al. International Journal of Systematic Bacteriology vol. 29 p. 92-101, 1979. 4. H. W. Horowitz, et al. Journal of Clinical Microbiology vol. 25 p. 1562-1563, 1987. 5. T. J. Nicholls, et al. The Veterinary Record vol. 109 p. 288, 1981. 6. M. L. Pineda-Castellanos, et al. Pathogens vol. 4 p. 210-228, 2015.

61 P 8. The evaluation of the activity of cepharanthine in human glioma multiforme (SNB-19) in vitro K. Cierluk1, J. Kulbacka2

1Faculty of Chemistry, Wroclaw University of Science and Technology, Wroclaw, Poland 2Department of Molecular and Cellular Biology, Wroclaw Medical University, Wroclaw, Poland Glioblastoma multiforme is one of the intracranial tumors leading to brain dysfunction. Currently, there are no effective therapies – commonly tumor resection, radiotherapy and chemotherapy are applied. The prognosis for its patients are not promising, which is why new therapies are being sought [1]. Cepharanthine (biscoclaurine) was isolated from the plant Stephania cepharantha Hayata. In Asia, Stephania is used to treat chronic and severe diseases. That wide application in medicine is very popular among scientists [2]. Biscoclaurine is an alkaloid which belongs to the group of bis-benzylquinoline compounds [3]. It is believed to have anticancer properties [4]. In order to investigate the effect of cepharanthine on glioblastoma multiforme cells, studies were performed in vitro using cell lines – SNB-19 (glioblastoma multiforme cells) and PC-12 (rat adrenal tumor cells induced to normal nerve cells by use of neural growth factor, NGF). Cepharanthine cytotoxicity assay was performed using MTT assay, T-type calcium channel and VDAC-1 mitochondrial pores labelling by the ABC immunocytochemical method, immunofluorescence labeling of reactive oxygen species, identification of death type and visualization of cepharanthine binding to VDAC-1 by ligand- protein docking. After analysis of the conducted studies, it appears that cepharanthine has an induction effect on the internal path of apoptosis by affecting the expression of VDAC-1 and stimulation of the production of reactive oxygen species. ACKNOWLEDGEMENTS The work was created as part of the activity of Student Research Group “Biology of Cancer Cell” at the Wroclaw Medical University (SKN No. K 148) and Statutory Funds of Department of Molecular and Cellular Biology REFERENCES: 1. J. Sokołowska, P. Sysa, M. Szmidt, K. Urbańska; „Glioblastoma multiforme – an overview”; Division of Histology and Embryology, Department of Morphological Science, Faculty of Veterinary Medicine, Warsaw University of Life Sciences-SGGW; 2014; 18(5); 307-312. 2. M. Baksalerska – Pazera, G. Niewiadomska; „NGF na szlaku, czyli przekazywanie sygnału troficznego w neuronie”; Instytut Biologii Doświadczalnej im. M. Neckiego PAN, Zakład Neurofizjologii, Warszawa; Kosmos – problemy nauk biologicznych; 2001; 50(1-2); 19-28. 3. K. Kono, J. A. Takahashi, T. Ueba, H. Mori, N. Hashimoto, M. Fukumoto; „Effects of combination chemotherapy with biscoclaurine-derived alkaloid (Cepharanthine) and nimustine hydrochloride on malignant glioma cell lines.” J. Neurooncol; 2002; 56(2); 101-108. 4. P. L. Schiff, Jr.; „ Bisbenzylisoqunoline Alakloids”; Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, Pennsylvania 15261; Journal of Natural Products; 1991; 54(3); 645-749.

62 P 9. Free-standing nanostructured films from amyloidogenic proteins scaffolded in Poly(vinyl alcohol) matrix

P. Cwynar, J. Olesiak-Bańska

Advanced Materials Engineering and Modelling Group, Faculty of Chemistry, Wroclaw University of Science and Technology, Wroclaw, Poland Amyloidogenic proteins in the aggregated form are related with neurodegenerative diseases. Simultaneously, they can form highly organized material, very promising in biomaterials field. Amyloids proteins possess unique mechanical properties with the Young’s modulus up to 6-7 GPA [1] that is much higher than proteins in native or in monomer forms. Their stiffness, resistance and natural perfect self-organization make them a very unique multifunctional material for biological, medicine and even electronic applications. Biofilm made of amyloid scaffolded on PVA matrix is a freestanding film that can be use as an artificial biomembrane but also as an electronic nanodevices, an optical waveguide and much more [2]. In this work we have successfully fabricated an ordered amyloids biofilm of lysozyme and insulin embedded in poly (vinyl alcohol) matrix. Morphology of the film was investigated with atomic force microscope and polarized light microscopy with full wave plate that highlighted the highly-organized domains. These domains were also studied with two-photon microscopy with and without fluorescence dye thioflavin T. All the techniques confirmed macroscopic and microscopic alignment of amyloid fibers. REFERENCES: 1. Knowles T.P.J., et al., “Nanostructured films from hierarchical self-assembly of amyloidogenic proteins” 2010. - 5: p. -. 2. Wei, G. et al., “Self-assembling peptide and protein amyloids: from structure to tailored function in nanotechnology” Chemical Society Reviews, 2017. 46(15): p. 4661-4708.

63 P 10. Diagnodent and VistaCam may be unsuitable for the evaluation of dental caries in archeological teeth P. Dąbrowski1, M. Kulus2, J. Grzelak3, T. Staniowski4

1Division of Normal Anatomy, Department of Human Morphology and Embryology, Wrocław Medical University, Wrocław, Poland 2Division of Histology and Embryology, Department of Human Morphology and Embryology, Wrocław Medical University, Wrocław, Poland 3Department of Oral Anatomy, Wrocław Medical University, Wrocław, Poland 4Department of Conservative Dentistry and Pedodontics, Wrocław Medical University, Wrocław, Poland One of the problems occurring in dental studies of historic and prehistoric populations is the appropriate assessment of early carious lesions. In such cases, the assessment may be imprecise or sometimes even erroneous, especially for initial stages of caries. These stages (in the form of white or brown discolouration) can be mistaken for non-pathological discolouration caused by environmental factors or taphonomic changes that can “mimic” early carious lesions. The aim was to compare the usefulness of fluorescence‐based, clinical caries detection systems (Diagnodent and VistaCam) for the assessment of carious lesions on archeological molars. The study material consisted of teeth from the Cemetery of St. Mary Magdalene in Wrocław, Poland. A sample of 178 permanent molars from 38 skulls were examined. Occlusal, mesial, distal, buccal, and lingual surfaces were assessed on either basically cleaned or sandblasted teeth. Six diagnostic methods were used to detect carious lesions: the visual classification of the International Caries Detection and Assessment System (ICDAS II), fluorescent methods (Diagnodent and VistaCam), X‐ray, cone beam computed tomography and histological sections. The sensitivity and specificity of the methods were determined using receiver operating characteristic (ROC) curves. Diagnodent and VistaCam yielded unsatisfactory specificity and sensitivity values. The area under curve (AUC) values for Diagnodent and Vistacam were lower than the those obtained for the ICDAS II classification. According to our results, in the case of archeological teeth, neither Diagnodent nor VistaCam can be regarded as a better diagnostic method than the ICDAS II visual classification of caries. ACKNOWLEDGEMENTS The authors thank prof. Barbara Kwiatkowska, Jacek Szczurowski, PhD and Dariusz Nowakowski, PhD from the Department of Anthropology, Wrocław University of Environmental and Life Sciences for sharing the study material. Also: to prof. Urszula Kaczmarek head of the Department of Conservative and Paediatric Dentistry, Medical University of Wrocław for sharing a Diagnodent Pen 2190 and a VistaCam iX. The research was financed by the Polish National Center of Science under the project “OPUS 13”, decision number: DEC-2017/25/B/HS3/02006.

64 P 11. Modifications of chemotherapy in cisplatin resistant human ovarian cancer M. Dębiński1, J. Kulbacka1, J. Bar2 J. Saczko1

1 Department of Molecular and Cellular Biology, Wrocław Medical University, 211 Borowska Str., 50-556 Wrocław, Poland 2 Department of Immunopathology and Molecular Biology, Wrocław Medical University, 211 Borowska Str., 50-566 Wrocław, Poland Ovarian cancer is the seventh most common cancer and the eight cause of death from cancer in woman. The disease typically presents at late stage when the 5-year relative survival rate is only 29% [1, 2]. Many of the patients dies because of low efficiency of second- and third-line chemotherapy [3]. Therefore there is a need to establish more effective treatment techniques. Several studies have shown promising results of combining cytostatics [4, 5] and calcium ions, [6, 7] with electroporation (EP) in treatment protocols in case of various cancers. The aim of the study was to investigate in vitro effectiveness of this methods in cisplatin resistant human ovarian cancer line OvBH-1. There was utilized nanosecond range electroporation protocol (10ns) with high intensity of the electric field (up to 10 kV/cm). The viability of cells was evaluated by MTT assay. Additionally, the impact of 17β-estradiol on efficacy of the protocols was examined. The obtained results indicated a promising enchanced anticancer effect of 17β-estradiol preincubation on the nanoelectrochemotherapy in ovarian cancer. ACKNOWLEDGEMENTS The work was created as part of the activity of Student Research Group “Biology of Cancer Cell” at the Wroclaw Medical University (SKN No. K 148) and Statutory Funds of Department of Molecular and Cellular Biology. REFERENCES: 1. J. Ferlay, et al. “Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012” International Journal of Cancer, vol. 136; p. E359-E386; 2015. 2. B.M. Reid, et al. “Epidemiology of ovarian cancer: a review” Cancer Biol Med., vol. 14; p. 9-32; 2017. 3. M. Miedzińska-Maciejewska, et al. “The modulation of multidrug resistance in ovarian cancer patients” Współczesna Onkologia, vol. 8; p.457-465; 2004. 4. B. Mali, et al. “Antitumor effectiveness of electrochemotherapy: A systematic review and meta- analysis” European Journal of Surgical Oncology, vol. 39; p. 4-16; 2013. 5. J. Saczko, et al. “The effectiveness of chemotherapy and electrochemotherapy on ovarian cell lines in vitro. Neoplasma” vol. 63; p. 450-455; 2016. 6. H. Falk, et al. “Calcium electroporation for treatment of cutaneous metastases; a randomized double- blinded phase II study, comparing the effect of calcium electroporation with electrochemotherapy” Acta Oncologica, vol. 53; p. 311-319; 2018. 7. A. Szewczyk, et al. “Calcium electroporation for treatment of sarcoma in preclinical studies” Oncotarget, vol. 9; p. 11604-11618, 2018.

65 P 12. A new molecularly imprinted polyaniline - based biosensor for detection of β-17 estradiol K. Drzozga1, J. Cabaj1 1Faculty of Chemistry, Wrocław University of Science and Technology, Wrocław, Poland One of the biggest threats to environmental protection is the presence of endocrine disrupting compounds (EDCs). This group includes polychlorinated dibenzodioxins, polychlorinated biphenyls, active substances contained in pesticides (biocides), phthalates and alkylphenols. In addition to these compounds, EDC also includes natural hormones and synthetic hormones. EDCs are factors that interfere with the production, release, transport, metabolism, action or elimination of natural hormones in the body. They are therefore substances that disturb the hormonal balance of organisms, therefore should be treated as dangerous substances. In addition to the negative impact on human hormone metabolism, EDCs also have a negative impact on aquatic organisms. These components accumulate in bottom sediments and are absorbed by aquatic organisms. The most common result is the feminization of male individuals and reproductive disorders [1]. Molecular imprinting polymers (MIPs) are artificially synthesized synthetic polymer sorbents. They exhibit selectivity for a given analyte or group of structurally similar analytes. This is due to the fact that during synthesis, binding sites specific for the substance to be detected are formed on their surface. MIPs are used as a substitute for antibodies, enzymes and other native biologically active structures in intermolecular recognition studies [2]. To construct an MIP-based sensor for the detection of β-17 estradiol, electrochemical synthesis of MIP in the estradiol and aniline environment was performed by cyclic voltammetry (CV). The constructed system was tested against various hormone concentrations by differential-pulse voltammetry (DPV). Researches were also carried on checking the influence of interfering compounds on the work of the sensor. As a result, a constructed platform is characterized by high selectivity and a wide linear range, and its high detection limit allows the determination of small concentrations of estradiol in the tested samples.

ACKNOWLEDGEMENTS The authors thank to Wrocław University of Science and Technology for financial support. REFERENCES: 1. H. Langauer-Lewowicka, et al. Environ. Medicine, 18, p. 7-11, 2015. 2. X. Zhang, et al. Sens. Actuators B., 200 p. 69-75, 2014.

66 P 13. Analogies in pathogenesis of Alzheimer’s disease and type 2 diabetes mellitus A. Dyba1, A. Kubis-Kubiak2, A. Piwowar2

1Student Science Club of Department of Toxicology, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, Wroclaw, Poland 2Departmentof Toxicology, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, Wroclaw, Poland Alzheimer’s disease is a neurodegenerative disease characterized by progressive cognitive and behavioral deficiency. Along with type 2 diabetes mellitus, a metabolic disorder depicted primarily by hyperglycemia and insulin resistance represents a considerable public health issue. Furthermore, both Alzheimer’s disease and type 2 diabetes mellitus are amyloidogenic diseases, with abnormal aggregation of amyloid-β peptide and islet amyloid polypeptide respectively contributing to cellular death and disease pathogenesis [1]. Recent studies have conceded common mechanisms partaking in pathogenesis of Alzheimer’s disease and type 2 diabetes mellitus on molecular and cellular levels. Markers of metabolic dysfunction exist also in Alzheimer’s disease, the most distinctive being insulin resistance [2]. In addition to the widely recognized role, insulin modifies neuronal activity, promotes synaptic plasticity and improves memory functions [3]. Insulin resistance, classically occurring in peripheral tissues in diabetes mellitus, has been revealed to be impaired in brains of Alzheimer’s disease patients. The mechanisms of brain disturbances include tumor necrosis factor-alpha mediated chronic inflammation, protein tyrosine phosphatase 1B hyperactivity, mammalian target of rapamycin signaling and atypical ganglioside metabolism, they seem to be distinctively akin in both diseases [4]. Those mechanisms result in, among others, insulin receptor impairment, synaptic plasticity and cognition decrease and endoplasmic reticulum stress. Molecular connections between irregular insulin metabolism in Alzheimer’s disease and diabetes mellitus present an ongoing challenge of innovative therapeutic applicability of anti-diabetic agents in a multimodal treatment of Alzheimer’s disease as they may be beneficial for cognition, synapse protection and insulin signaling [5]. REFERENCES: 1. S.M.de la Monte. “Contributions of Brain Insulin Resistance and Deficiency in Amyloid-Related Neurodegeneration in Alzheimer’s Disease” Drugs.72, p. 49-66, 2012. 2. L.S.S.Ferreira, et al. “Insulin Resistance in Alzheimer’s Disease” Front Neurosci. 12,:p. 830, 2018, 3. H.J.Lee, et al. “Diabetes and Alzheimer’s Disease: Mechanisms and Nutritional Aspects” Clin Nutr Res. 2018; 7(14): 229-240. 4. M.N.N. Vieira, et al. “Connecting Alzheimer’s disease to diabetes: Underlying mechanisms and potential therapeutic targets” Neuropharmacology136(Pt B), p. 160-171, 2018. 5. S. Takeda, et al. “Molecular mechanisms linking diabetes mellitus and Alzheimer’s disease: beta- amyloid peptide, insulin signaling, and neuronal function” Mol Biosyst.7, p. 1822-1827, 2011.

67 P 14. Comparison of the activity of commonly-used antiseptics against methicillin-resistant and methicillin-sensitive Staphylococcus aureus biofilm formed in various culturing conditions K. Dydak1, J. Paleczny1, M. Oleksy1, A. Junka1 , M. Bartoszewicz1

1Department of Pharmaceutical Microbiology and Parasitology, Wroclaw Medical University, Wroclaw, Poland Introduction: Staphylococcus aureus is a widespread microorganism responsible for hospital and community-acquired infections including these in chronic wounds. Ability to form biofilm structure is one of the numerous virulence factors of aforementioned bacterium. It prevents majority of antibiotics from penetration and contributes to increased tolerance also against antiseptics, i.e. locally-administered antimicrobials commonly used, among others, to prevent and treat wound infections. Aim: To compare activity of four antiseptics against methicillin-resistant and methicillin- susceptible S. aureus (MRSA and MSSA, respectively) biofilm in two types of culture media. Materials and methods: 10 strains of Staphylococcus aureus (including reference ATCC 6538 (MSSA) and ATCC 33591 (MRSA) and 8 clinical S. aureus strains were used for experimental purposes. Strains were cultured in tryptic soy broth (TSB) or in the defibrinated sheep blood (SB). The antibiofilm-activity of the following antibacterial agents: octenidine dihydrochloride (OCT), polyhexamethylene biguanide (PHMB), povidone iodine (PVP-I) and ethacridine lactate (EL) were examined by means of the Minimum Biofilm Eradication Concentration (MBEC) method. Results: The average MBEC of OCT and PVP-I for MSSA cultured in TSB were respectively: 75 mg/l, 843,75 mg/l; for MRSA: 50 mg/l, 937,5 mg/l. All strains were resistant to EL and PHMB within tested range of concentrations. The average MBEC of OCT and PHMB for MSSA strains cultured in SB were respectively: 28,1 mg/l, 8,59 mg/l and for MRSA: 51,6 mg/l, 12,5 mg/l. 1 MSSA and 3 MRSA were resistant to EL, 3 MSSA and all MRSA strains were resistant to PVP-I within tested range of concentration in this experimental settings. Conclusion: Tryptic soy broth ensures higher tolerance of examined Staphylococcus aureus strains to anti-biofilm activity of octenidine dihydrochloride, polyhexamethylene biguanide and ethacridine lactate than the defibrinated sheep blood does. The inverse tendency is observed in anti-biofilm activity of povidone iodine. Tolerance of examined S. aureus strains to PVP-I is higher in the SB than in the TSB.

Research was performed by means of statutory research ST.D230.18.008

68 P 15. The evaluation of the activity of kavain in human glioma multiforme (SNB-19) in vitro M. Geneja1, J. Kulbacka2

1Faculty of Chemistry, Wroclaw University of Science and Technology, Wroclaw, Poland 2Department of Molecular and Cellular Biology, Wroclaw Medical University, Wroclaw, Poland Kavain is a natural substance derived from a plant and has promising antiproliferative properties towards different types of tumor cells [1]. The evaluation of the activity of kavain has been performed at Wroclaw Medical Univeristy in the laboratory of the Department of Molecular and Cellular Biology. Glioblastoma multiforme cells were selected as a research object to determine kavain impact, because of high malignancy of this cancer and difficult and challenging treatment. Experimental studies have been conducted on human brain tumor cell line (SNB-19) with the use of such laboratory methods as MTT assay, immunohistochemistry and immunofluorescence staining and flow cytometry. The same tests have been done on a model of normal nerve cells (PC-12 cell line stimulated by NGF factor) and served as a control studies. The cellular expression of mitochondrial and membrane channels (exampled by aquaporin 4 [2], T type calcium channel [3] and Maxi-K potassium channel [4]) has been examined after incubation with kavain. The obtained results indicate the induction of apoptosis by the intrinsic pathway in both of the studied cell lines. ACKNOWLEDGEMENTS The work was created as part of the activity of Student Research Group “Biology of Cancer Cell” at the Wroclaw Medical University (SKN No. K 148) and Statutory Funds of Department of Molecular and Cellular Biology REFERENCES: 1. M. Gregory, G. Steiner, “The correlation between Cancer Incidence and Kava Consumption” Hawaii Medical Journal, vol.59, p. 420-422, 2000. 2. T. Ding, Y. Zhou, K. Sun, et al., “Knockdown a Water Channel Protein, Aquaporin-4,Induced Glioblastoma Cell Apoptosis” Plos One, p. 1-9, 08. 2013. 3. N. N. Phan, C. Y. Wang, C.-F. Chien, et al., “Voltage-gated calcium channels: Novel targets for cancer therapy” Oncology letters, p. 2059-2074, 2017. 4. I. Kazama i Y. Maruyama, “Inner mitochondrial maxi-K+ channels in neonatal renal tubular cells: novel therapeutic targets to control apoptosis” Medical Hypotheses, p. 800-801, 2012.

69 P 16. Antiproliferative efficacy of lipid nanoparticles prepared from anisic/veratric acid-phospholipid conjugates towards leukemia cells line MV4-11 1 1 1 2 2 3 A. Gliszczyńska , N. Niezgoda , M. Czarnecka , M. Świtalska , J. Wietrzyk , E. B. Souto 1Department of Chemistry, Wroclaw University of Environmental and Life Science, Wroclaw, Poland 2Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland 3Department of Pharmaceutical Technology, University of Coimbra (FFUC), Coimbra, Portugal The lymphatic system plays a crucial role in cancer diseases. Many cancers origine from this system e.g. lymphoma, myeloma, leukemia or Kaposi's sarcoma, and clinical observations indicate that also other types of tumors are spread in the organism via lymphatic system. Therefore, logical seems to be employment this system for distribution of substances with chemopreventic and cytotoxic activity in the body, what is possible for lipophilic compounds such as phospholipids. Methoxybenzoic acids (OMe-BA) are known from their broad range biological activity, also as the agents able to induce apoptosis of cancer cells. It has been proven that some OMe-BA e.g. anisic and veratric acids covalently bounded with phosphatidylcholine as phospholipid conjugates exhibit significantly higher antiproliferative activity than their free form. Moreover, for this type of biomolecules, good strategy for their effective delivery to the target cells is to use them in the form of nanoparticle. Among them the most interesting are solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLCs), which are stable in the blood system, guarantee slow release in the body of the active substance. Their production is also supported by the fact of relatively low price and easy technology of production in the industry. In our report, heterosubstituded phosphatidylcholines enriched with anisic/veratric acid in sn-1 or sn-2 positions were used as biologically active surfactants in preparation of two types of lipid nanoparticles SLN and NLC respectively. Cytotoxic effect of developed delivery systems for anisic and veratric acids was next evaluated on human leukemia cancer cell line MV4-11.

70 P 17. Square wave voltammetry (SWV) using a gold disc microelectrode – the study of ephedrine oxidation O. Gładysz1, P. Skibiński1

1Department of Analytical Chemistry, Wroclaw Medical University, Wroclaw, Poland [email protected] Ephedrine (1R,2S)-2-(methylamino)-1-phenylpropan-1-ol is an alkaloid which is naturally found in Ephedra species. As a sympathomimetic agent acts as α and β-adrenergic agonists, stimulates the central nervous system. Besides therapeutic indications (bronchodilator), ephedrine is used as a doping agent in sports (improves athletic performance and accelerates weight loss). World Anti-Doping Agency included ephedrine on the list of banned substances during the competition, in group of non-specified stimulants [1]. There are several methods for its determination (e.g. chromatographic methods, coupled to mass spectroscopy, capillary electrophoresis techniques) but these methods are relatively time-consuming and require sample pre-treatment. Research methods that are cost-effective and provide the possibility of on-line measurement are electrochemical methods. They are efficiently used to determine psychoactive substances [2] and to control quality, stability, bioavailability of drugs [3]. An alternative tool to conventional size electrodes are microelectrodes with dimensions within a µm range and the numerous advantages (small currents in the range of nA, increased current density, higher ratio of faradaic current to capacitance current, improvement in signal to noise ratio, possibility in vivo measurements) [4]. Square wave voltammetry (SWV) and a gold microelectrode (diameter 25 µm) was applied to study the ephedrine oxidation in solutions made of pharmacopoeia standard substance, in acidic pH. The dependence Ipeak = f(Cephedrine) is linear. Calibration curve, detection (LOD) and quantification (LOQ) limits were calculated in fixed experimental condition. SWV is one of voltamperometric methods of high analytical sensitivity and numerous advantages [5]. The results are the initial attempt in development an alternative, direct, simple and fast method for ephedrine determination, which could replace the time-consuming and cost-generating methods used nowadays for drug quality or anti-doping control. REFERENCES: 1. www.wada-ama.org/en/content/what-is-prohibited/prohibited-in-competition/stimulants (access 04.02.2019) 2. G. B. Slepchenko, et al. J. Anal. Chem. vol. 72(7) p. 703-709, 2017 3. N. A El-Maali, Bioelectrochem. vol. 64(1) p. 99-107, 2004. 4. K. Stulík, et al. Pure Appl. Chem, vol. 72 (8) p. 1483-1492, 2000). 5. V. Mirceski, et al. Electroanal. Vol. 25(11) p. 2411-2422, 2013

71 P 18. Impact of patients serum with disturbed carbohydrates metabolism on the PC12 cell line viability – preliminary studies N. Głogowska1, A. Rorbach-Dolata2, D. Bednarska-Chabowska3, A. Piwowar2

1Students' Scientific Society at the Department of Toxicology, Wroclaw Medical University, Wroclaw, Poland 2Department of Toxicology, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, Wroclaw, Poland 3Department of Angiology, Hypertension and Diabetes, Faculty of Postgraduate Medicine, Wroclaw Medical University, Wroclaw, Poland Introduction. Disorders of carbohydrate metabolism are considered to be the cause of many civilization diseases. Currently there are theses linking the abnormal metabolism of glucose with neurodegenerative diseases, giving them the title of neurometabolic diseases. The aim of the study was to evaluate the effect of biological material (human serum) from patients with diagnosed carbohydrate metabolism disturbances on the survival of PC12 cells. Material and methods. The experiment used undifferentiated cell line PC12, commonly applied in cytotoxicity studies. The cells were incubated for 48 hours in standard conditions of medium (with addition of appropriate agents), temperature and atmosphere with serum (20% v/v) with high or normal glucose and insulin level. Metabolic activity of PC12 cells was assessed by MTT test and percentage of survival cells (SP%) were calculated. Moreover, laboratory parameters such as glucose and insulin level in biological material were also evaluated. Results. The results indicate that SP% of PC12 cells after incubation with human serum is lower compared to the control group. In addition, cell line survival increases with increased glucose levels, but decreases with increased insulin levels in specific groups. Additionally some connection with sex was observed – the SP% of PC12 cells incubated with woman serum was lower than in men. Conclusions. Glucose and insulin disorders negatively affect the survival of PC12 cells. Hyperinsulinemic states are more cytotoxic to PC12 than hyperglycemic states. Observed reduced survival of PC12 cells after incubation with serum from women can be probably associated with a different hormonal sex status. REFERENCES: 1. De La Monte S. “Alzheimer’s Disease Is Type 3 Diabetes – Evidence Reviewed” J. Diabetes Sci. Technol., 2008, 22(6), 1101-1113. 2. Chen M., Zheng H., Wei T. “High glucose-induced PC12 cell death by increasing glutamate production and decreasing methyl group metabolism” BioMed Res. Int., 2016. 3. Oleśkowa-Florek W. “Ocena parametrów zaplanych w surowicy chorych dializowanych i wpływu tej surwicy na komórki śródbłonka w hodowli in vitro” rozprawa doktorska, 2013 4. Rorbach-Dolata A., Piwowar A. “Adverse effect of the disturbances of glycemia and insulimemia on model PC12 cells – Preliminary report, Pediatr Endocrinol” Diabetes Metab., 2017, 3(4), 174-180.

72 P 19. Kinetics of bovine insulin fibrillation M. Grelich, J. Olesiak-Bańska

Advanced Materials Engineering and Modelling Group, Faculty of Chemistry, Wroclaw University of Science and Technology, Wroclaw, Poland Some proteins in mildly destabilising conditions undergo misfolding into aggregate structures known as amyloid fibrils. Mentioned assemblies prompt a challenging research interest since they are related to some neurodegenerative diseases, including Alzheimer’s and Parkinson’s disease [1, 2]. Fibrils obtained from bovine insulin present straight, unbranched morphology, can reach several microns in length and 3-15 nm width [3]. In vitro detection of these assemblies can be performed using Thioflavin T (ThT) dye which induces characteristic fluorescence upon binding to the fibrils [4]. In our studies, standard physicochemical techniques were used to monitor amyloid growth kinetics of bovine insulin. Obtained data reveal that ThT fluorescence kinetics presents a sigmoidal curve, in which lag, growth and plateau phases can be distinguished. We compared the results to the growth of fibrils without ThT and observed a change in the duration of each growth step. Then we performed atomic force microscopy imaging of the fibrils morphology in different time points. It is evidenced that ThT assay and time-lapse AFM imaging give a good insight into fibrillation process, with the understanding how the fluorescent probe affects this process. ACKNOWLEDGEMENTS We gratefully acknowledge funding from First Team Programme: "NONA - nonlinear optics, nanoparticles and amyloids". REFERENCES: 1. M. Stefani, International Journal of Molecular Sciences, 9, 2515-2542 (2008). 2. M.I. Smith, et al. Soft Matter, 8, 3751-3755 (2012). 3. J. Brange, et al. Journal of Pharmaceutical Sciences, 86, 517-525 (1997). 4. M. Groenning, Journal of Chemical Biology, 3, 1-18 (2010).

73 P 20. Emulsomes as a bioactive compounds delivery system for pancreatic ductal adenocarcinoma A. Grzeszczak, M. Zaremba-Czogalla, J. Gubernator

Department of Lipids and Liposomes, Faculty of Biotechnology, University of Wroclaw, Poland Pancreatic cancer is the fourth major cause of cancer death worlwide. The most common type of this cancer is pancreatic ductal adenocarcinoma (PDA). Pancreatic cancer is a highly lethal disease which very often does not cause any symptoms until the cancer reaches an advanced and commonly metastatic stage. Therefore the survival rate of patients is poor. The best treatment option depends on the disease stage and currently standard treatments are surgical resection (when feasible), chemoradiation and chemotherapy with combination of synthetic drugs. The limitation of effective treatment of PDA is a tumor structure which is characterized by very high levels of dense fibrotic stroma and insufficient vasculature [1]. Nowadays attention is focused on biological active food delivered substances with well documented anticancer properties. One of these compounds is phenethyl isothiocyanate (PEITC) which naturally occurs in cruciferous vegetables such as broccoli, watercress, Brussels sprouts, cauliflower, kale. PEITC is released from glucosinolates as an enzymatic hydrolysis product. Myrosinase an enzyme is activated by chewing or cutting the vegetables. Phenethyl isothiocyanate has demonstrated good anticancer, chemopreventive and chemotherapeutic activity. The limitation of PEITC to be considered as a drug is its physicochemical properties. This compound is hydrophobic, non-water soluble, highly volatile with low oral bioavailability. Furthermore PEITC is metabolized rapidly by glutathione conjugation then excreted in the urine as the mercapturic acid [2]. The main aim of the present work was to investigate anticancer activity of emulsomal formulations of phenethyl isothiocyanate on tested pancreatic cancer cell lines, ASPC-1 and BXPC-3. The efficiency of PEITC encapsulation was around 50% for all formulations. This study suggests that emulsomes could be a promising system able to encapsulate and transport expected substances to the tumor tissue. REFERENCES: 1. E.G. Chiorean, A Coveler. “Pancreatic cancer: optimizing treatment options, new, and emerging targeted therapies” Drug Design, Development and Therapy vol 9. 3529-3545, 2015. 2. J.V Higdon, B Delage, D.E Williams, R.H Daswood. “Cruciferous vegetables and human cancer risk: epidemiologic evidence andmechanistic basis” Pharmacological Research vol.55. 224-236, 2007.

74 P 21. Antioxidant activity of craft beers vs. commercial beers J. Harasym1,2, R. Olędzki1,2, M. Spanu3, U. Kaim1,2, L. Bogacz-Radomska1,2, A. Orkusz1,2

1. Adaptive Food Systems Accelerator – Science Centre, Wrocław University of Economics, Poland; 2. Department of Biotechnology and Food Analysis, Institute of Chemistry and Food Technology, Wrocław University of Economics, Poland; 3Agrary Department, University of Sassari, Italy Beer usually is not considered as source of antioxidants, and typically contains mainly alcohol and extract being results of fermentation process. However, recently rising interest in craft beer production led to significant increase in non-commercial products offer, which quality in terms of nutrients content can be far different from commercial offer. As beers are manufactured in specific types called “styles” are restricted by recipes and the ingredients which allow brewing distinct beers. But significant difference in industrial scale manufacturing and craft brewing can result in variation of chemical contents of beer. Additionally less rigorous approach to recipe in craft brewing and far more free fermentation time regime create the opportunity to change significantly the chemical content of manufactured beer. The main objective of this study were the assessment and the comparison of antioxidant activity of 9 Indian Pale Ale style beers manufactured by craft breweries and big beer companies. The assessment was proceeded using Folin-Ciocalteu, ABTS, DPPH, FRAP and flavonoids methods. The total polyphenol content expressed as gallic acid equivalent was significantly higher in craft beers and their content was not correlated with sugars content, which means that activity measured with Folin-Ciocalteu reagent was not biased by reducing sugars presence. In contrary higher levels of flavonoids were observed for commercial beers than craft ones. DPPH, ABTS and FRAP methods confirmed the higher antioxidant activity trend for craft beers although no for each beer the predominance was obvious. Summarizing, the advantages of craft manufacturing of beer in case of higher antioxidant activity seems to be confirmed.

75 P 22. Buckwheat flour antioxidant activity after ultrasound treatment J. Harasym1,2, E. Satta3, U. Kaim1,2, L. Bogacz-Radomska1,2, A. Orkusz1,2, R. Olędzki1,2

1. Adaptive Food Systems Accelerator – Science Centre, Wrocław University of Economics, Poland; 2. Department of Biotechnology and Food Analysis, Institute of Chemistry and Food Technology, Wrocław University of Economics, Poland; 3 Department of Agricultural and Food Science and Technology, University of Bologna, Italy Buckwheat (Fagopyrum esculentum Moench) is a non-cereal plant classified as pseudocereal due to large achene containing a significant amount of starch. As naturally gluten-free plant is a perfect raw material for formulation of gluten-free products although its specific taste and aroma can hinder its widespread usage. Buckwheat is also a medicinal plant with a specific application in traditional medicine. Buckwheat achenes are an important source of antioxidant compounds mainly polyphenols which can contribute to the total antioxidant status of buckwheat derived products. Although specific aroma, especially pronounced after thermal treatment, may restrict its application in food products, buckwheat flour can be used as a thickener in many semi-liquid products. To antioxidant activity of flour can improve the stability of oil emulsions, prolong the shelf life and restrict usage of preservatives. Therefore, the antioxidant activity of raw material is an important factor not only contributing to nutritional value but also facilitating products design and manufacturing. The main objective of this study was the assessment of antioxidant activity of buckwheat flours made from dehulled buckwheat achenes subjected to ultrasound processing in 40 kHz in different solid: liquid ratios (1: 10, 1:5, 1:2,5). The total polyphenols content both soluble and insoluble was assessed with Folin-Ciocalteu reagent while scavenging activity of antioxidant compounds was evaluated versus 1,1-diphenyl-2-picrylhydrazyl radicals. The total polyphenols content expressed as gallic acid equivalent was significantly changed in relation to solid: liquid ratio between buckwheat grain and water presenting a rising trend with decreasing ratio. However, antioxidant activity was the highest for the sample treated in ratio 1:5.

76 P 23. Visualization of Granzyme B using activity-based probe T. Janiszewski1, S. Kołt1, D. Kaisermann2, N. Bovenschen3, S. Snipas4, G. Salvesen4, M. Drąg1, P. Kasperkiewicz1

1Department of Bioorganic Chemistry, Wroclaw University of Science and Technology, 50-370 Wroclaw, Poland 2Monash Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton VIC 3800, Australia 3UMC Utrecht, 3584 CX Utrecht, Netherlands 4NCI-designated Cancer Center, Sanford-Burnham Prebys Medical Discovery Institute, 92037 La Jolla, USA Natural killer (NK) cells and cytotoxic T-lymphocytes (CTLs) are effector cells in the innate immune system involved in pathogen and cancer cells killing. Granzymes (Grs) is a group of serine proteases produced in the NK and CTLs and stored in the granules in the active form [1]. There are five granzymes in humans (GrA, GrB, GrH, GrK and GrM), however majority of studies focuded only on GrA and GrB, therefore other Grs are called orphan enzymes. Despite years of intensive studies on Grs family, their function and their exact location remain imprecise [2]. So far studies on Grs have been based on antibody-related techniques, that do not allow to distinguish between active and total number of Grs. Also, GrB antibody was found to be unselective and able to label GrA. Therefore, the ultimate goal of our research was to find a set of a molecules that will allow for GrB investigation. To date, GrB substrate specificity profile was determined only with limited number of amino acids, therefore in our work we used a set of moderate techniques that incorporates a wide range of unnatural derivatives to increase substrate specificity and potency of substrates/inhibitors. Only a few research groups investigated selective functional activity-based probes for Grs, however the activity and the selectivity of the molecules were not sufficient to perform biological assays on Grs with these molecules. Facing this problem, we have proposed and designed a new tools with unique sequences for Granzyme B analysis: substrate, inhibitor, covalent ABP and quenched probe, that possess high selectivity ratio and potency. In addition, we confirmed the utility of our molecules to GrB investigation on both a purified enzyme and in cells. ACKNOWLEDGEMENTS This work was supported by HOMING Programme, a Grant Project of Foundation for Polish Science, funded by the European Union under agreement No. 2016-3/24. PK is supported by Ministry of Science and Higher Education in Poland and by L’Oreal Foundation stipend For Women in Science. REFERENCES: 1. Ley T.J., et al. Immunity vol.26 p.798-811, 2007. 2. Craik C.S., et al. Chem Biol vol.12 p.567-577. 3. Hudig D., et al. Mol immunol vol.33 p.615-623

77 P 24. Interaction of Cu(II) ions with ANP – initial studies A. Jarmołkiewicz, A. Pieniężna, J. Brasuń Department of Inorganic Chemistry, Wrocław Medical University, Borowska 211A, 50-552 Wrocław, Poland ANP (artial natriuretic peptide) belongs to the family of natriuretic peptides and was investigated more than 20 years ago. It is secreted by the cardiac atrial cardiomyocytes and has a natriuretic, diuretic and hypotensive effects. This peptide hormone is very important in diagnosis and therapy. ANP protects body against the development of hypertension and excessive increase volume of the fluid space [1-3]. The active ANP hormone consists of 28 amino acids. In our studies we analyzed modified short cyclic fragment of this peptide, which contains 17 amino acids and a disulfide bridge between cysteine residues. Described molecules has also two histidine moieties in its sequence and due to this it could potentially bind Cu (II) ions [1, 3]. Cu (II) ions play an important role in biological systems including oxidation, electron transfer and dioxygen transport. In result of these processes they bind to peptides or proteins. Formed complexes could be potential precursors of drugs in theraphy in many diseases [4, 5]. Initial studies of coordination properties between ANP analogue and Cu (II) ions was conducted using various analytical methods such as UV-vis spectroscopy and CD spectroscopy. REFERENCES: 1. D. Pakuła, et al., Pol Journal Endocrinol, vol. 58, p. 364-374, 2007 2. K. Pandit, et al., Indian J Endocrinol Meatb., vol. 15, p. 345-353, 2016 3. H. Jerczyńska, Z. Pawłowska, Postępy Biochemii, vol. 54, p. 35-42, 2008 4. Z. Wu, et al., J Am Soc Mass Spectr, vol. 21, p. 522-533, 2010 5. A. Kotynia, et al., Int J Pept Res Ther, vol. 23, p. 431-439, 2017

78 P 25. African Swine Fever propagation in Europe – modelling perspective A. Jarynowski1, V. Belik2

1Interdisciplinary Research Institute in Wrocław, Poland 2Institute for VeterinaryEpidemiology and Biostatistics, Freie Universität Berlin, Germany African Swine Fever (ASF) is a viral infection which causes acute disease in domestic pigs and wild boar. Although the virus does not cause disease in humans, the impact it has on the economy, especially through trade and farming, is substantial causing more than one billion EUR yearly losses in Europe. Recent rapid propagation of the (ASF) from East to West of Europe (around 200km per year by 'jump wave' and around 20km per year by “diffusive wave”) encouraged us to prepare risk assessment for Poland. We developed a novel multilayer network model (incorporating domestic pig, wild boars and human layers) for epidemic spread of the African Swine Fever (ASF) in Poland and Eastern Europe which received international attention [1,2]. This modelling approach allows to understand the spreading mechanism, forecast of the future spread and to devise efficient control measures. The results are also helpful in the analysis of the spread of ASF in further European countries, which is a very urgent issue, as ASF already appeared in Belgium, thus affecting the Western Europe as well. ACKNOWLEDGEMENTS The authors thank CA15109 (COSTNET) and CA15116 (ASF-STOP) REFERENCES: 1. https://www.bloomberg.com/news/articles/2018-09-17/swine-fever-mystery-threatenschina-s-128- billion-pork-industry 2. https://www.topagrar.pl/articles/afrykanski-pomor-swin-asf/asf-za-trzy-lata-wwielkopolsce/

79 P 26. Pole on the appointment with the Dr Google- analysis of the phenomenon of online searching information about health J. Jeżewska1 , Z. Sycz2 , M. Sępek3, J. Barański4 1 Department of Public Health, Division of Medical Social Sciences, Wrocław Medical University, Bartla 5, 51-618 Wrocław, Poland 2 Department of Biology and Medical Parasitology, Wrocław Medical University, Mikulicza-Radeckiego 9, 50-367 Wrocław, Poland 3 Department of General, Minimally Invasive and Endocrine Surgery, Wrocław Medical University, Borowska 213, 50-556 Wrocław, Poland 4 Department of Humanistic Sciences In Medicine, Wrocław Medical University, J. Mikulicza - Radeckiego 7, 50-368 Wrocław, Poland The popularity of using the Internet has been increasing year by year [1-3]. At the end of 2018 the number of Internet users reached 28 million Poles [4]. In 2005 online health services were visited by approx. 2 million Poles (currently almost 13 million) [5]. The largest group of Internet users are people aged 25-34 [2, 6]. In the age range 15 to 34 there are much more women [2]. Internet users, in comparison to the general population in Poland, are better educated [2]. It’s difficult to distinguish reliable information. The problem is particularly important, because at the moment of health deterioration 69% of Internauts look for online information about their ailments and how to cope with them [7]. 14% of Poles treat themselves, relying on information gathered from the Internet network 7. The use of unverified health information may result in danger to life or health [8]. The aim of the study was to analyse the phenomenon of searching the web for health information in Poland depending on the demographic characteristics of Internet users. The study is a review of articles and reports describing the Internet use for collecting information on health.

Characteristics Behaviour care for their own health 9 expand or verify online the information Women 4 7 interested in health and lifestyle obtained from doctors determine themselves the method of believe that everyone is responsible for Young people 9 treatment, often independently basing on their health 7 information found on the web stress the value of health 9 often expand or verify online information High educated 7 most willing to use the Internet for health obtained from doctors and change people purposes 3,8 recommendation 7

1. There is a high probability that the phenomenon of searching health information online will increase year by year. The most involved and more often using health online services are women, young and better educated people who are willing to self-heal. They are more exposed to risky self-care. 2. It would be advisable to create tools which would allow people interested in health issues to obtain confirmed and based on current knowledge information on the Internet and to support eHealth literacy8. REFERENCES: 1. GUS. “Społeczeństwo Informacyjne w Polsce w 2018 r.”, 2018. 2. IAB Polska. Raport Strategiczny. INTERNET 2017/2018.; 2017. 3. Fiksdal, A. S., Kumbamu, A., Jadhav, A. S., Cocos, C., Nelsen, L. A., Pathak, J., & McCormick JB. “Evaluating the Process of Online Health Information Searching: A Qualitative Approach to Exploring Consumer Perspectives” J Med Internet Res. 2017;16(10). 4. Polskie Badania Internetu. Polscy internauci w grudniu 2018. 2019. Accessed January 27, 2019. 5. Polskie Badania Internetu. Internetowe Serwisy o Zdrowiu Zawartość i Popularność Serwisów, Profil Użytkowników.; 2016. 6. CBOS. Korzystanie z Internetu.; 2018. 7. CBOS. Zdrowie Online. Warszawa; 2016. 8. Waszak P, Kasprzycka-Waszak W. “Rola Internetu i Web 2.0 w zdrowiu publicznym-krótki przegląd podstawowych narzędzi oraz ostatnich polskich doświadczeń” Hygeia Public Heal. 2018;53(3):213-220. 9. CBOS. Zdrowie i Prozdrowotne Zachowania Polaków. Warszawa; 2016. 80 P 27. The isothiazole derivaitves with anticancer activity I. Jęśkowiak1, M. Mączyński1, J. Trynda2, J. Wietrzyk2, S. Ryng1

1 Department of Organic Chemistry , Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, Borowska 211a, 50-556 Wroclaw, Poland 2 Laboratory of Experimental Anticancer Therapy , Polish Academy of Sciences, Institute of Immunology and Experimental Therapy, Weigla 12, 53-114 Wroclaw, Poland [email protected] After cardiovascular diseases, cancers are recognized as the second most common reasonof death in USA and Europe [1]. The problems such as toxicity and drug resistance have stimulatedan intense demand and research efforts for the discovery of new and novel antitumor agents [2]. The isothiazole ring has been associated with a broad spectrum of pharmacological properties as an anticancer agent. 26 new derivatives of 5-hydrazine-3-methylisothiazole-4-carboxylic acid were obtained. Chemical structure was carried out using mass spectrometry (MS), proton nuclear magnetic resonance spectroscopy (1HNMR) and infrared spectroscopy (IR). The antiproliferative activity was examined in the methylthiazolyldiphenyl-tetrazolium bromide test (MTT). 13 compounds caused the high inhibition of the proliferation of human biphenotypic B cell myelomonocytic leukemia MV4-11. For 10 compounds IC50 values were designated but they were poor. For the next 4 compounds IC50 values were not determined. The anticancer activity of this group of isothiazole derivatives may be associate with the presence of –CH=N- (Schiff’s base) moiety and presence of substituted phenyl ring in position 5 of the isothiazole ring. Summarising, the synthesized isothiazole compounds are expected to exhibit anticancer activities and could be studied further as potential anticancer drugs. The result of this work is claimed by Polish patent application nr P.426618. The work was financed by Wroclaw Medical University supervisory research project for Young Scientists registered in the university records under the number STM.D090.17.003. REFERENCES: 1. D. Tewari et al. Food and Chemical Toxicology, 123, 522-535, 2019. 2. M. K. Ediriweera et al., Journal of Applied Toxicology, 39, 38-71, 2019.

81 P 28. Assessment of the activity of lactic acid dehydrogenase and prognosis in patients with lung cancer M. Jurczyk1, M. Śliwińska-Mossoń2, Ł. Trembecki3, H. Milnerowicz2

1SKN at Department of Biomedical Environmental Analyzes, Medical University of Wroclaw, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw, Poland 2Department of Biomedical Environmental Analyzes, Medical University of Wroclaw, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw, Poland 3Department of Radiotherapy, Lower Silesian Oncology Center, Wroclaw, Poland Lactate dehydrogenase (LDH) is a cytoplasmic enzyme from the class of oxidoreductases. Catalyzes the reversible reaction of lactate oxidation to pyruvate occurs under anaerobic conditions, such as intratumoral environment, this causes higher LDH activity in malignant tumors [1]. An appropriate combination of different polypeptide chains determines the presence 5 isoenzymes [2]. The even small damage can increase the plasma enzyme activity [3]. The last studies investigate the relationship between baseline serum LDH and survival of patients suffering from small cell lung cancer (SCLC). Among patients with an abnormally elevated LDH activity, those who had relatively higher LDH activity (>370 U/L) would have a poorer survival than those with moderately elevated LDH activity (245–370 U/L). Results indicated that patients with an abnormal elevation LDH activity would have a 1.45 times higher risk of death than those with normal LDH activity [4]. In the next study, also higher pretreatment LDH activity was significantly associated with an increased risk of overall mortality in patients with lung cancer [5]. In the other study were tested for LDH5 expression by immunohistochemistry using a polyclonal LDH5 antibody. 89.5% of non small cell lung carcinoma (NSCLC) revealed LDH5 expression whereas LDH5 expression was not detected in non neoplastic lung tissues LDH5 is overexpressed in NSCLC and could hence serve as an additional marker for malignancy [6]. The results suggested that higher pretreatment LDH activity and overexpression LDH5 was associated with worse overall survival in patients with lung cancer. The findings may assist future research on anticancer therapy by targeting LDH and help predict prognosis in lung cancer patients. REFERENCES: 1. C. Burtis, et al. Saunders Publishing Elsevier, St. Loius, USA. 321-322, 2008. 2. K. Świderek, et al. Arch. Biochem. Biophys, 505, 33-41, 2011. 3. S. Farugi, et al. Clin. Respir. J, 6, 81-87, 2012. 4. L. Zhou, et al. Journal of Thoracic Disease 10, 1043-1049, 2018. 5. T. Deng, et al. Medicine (Baltimore), 97, 12524, 2018. 6. G. Kayser, et al. Diagn Pathol, 1746-1596-5-22, 2010

82 P 29. Metalloestrogens as a new but important player in male infertility problem K. Jurkowska1, E. M. Kratz2, E. Sawicka3, A. Piwowar3

1Doctoral Student and Student Science Club of Department of Toxicology, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, Wroclaw, Poland 2Department of Laboratory Diagnostics, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, Wroclaw, Poland 3Department of Toxicology, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, Wroclaw, Poland In highly developed countries, there is an evidence trend of reducing the birth rate. One of the causes of this negative phenomenon is the decreasing reproductive potential of men. Chronic exposures on different impurities, the impact of environment, diet or drug on male reproductive health are indicated as important agents [1]. Scientific data confirmed that many compounds in the environment have been shown capable of binding to cellular estrogen receptors and then mimicking the actions of physiological estrogens. The widespread origin and diversity in chemical structure of these environmental estrogens is extensive but to date such compounds have been organic (phenolic or carbon ring structures of varying structural complexity) [2]. They have been called xenoestrogens. However, recent reports also showed the ability of certain metal ions to bind the estrogen receptors and give rise to estrogen agonist responses. It has resulted in the realisation that environmental estrogens can also be inorganic and such xenoestrogens have been termed metalloestrogens [3]. Their role in the disturbances of women's health, especially some xenoestrogens in breast or ovarian cancer development is well known [4], but still not enough is known about their role in male health [5]. Especially little is known about participation of metalloestrogens in male infertility. The following metals were capable of binding to cellular estrogen receptors and then mimicking the actions of physiological estrogens: aluminium, antimony, arsenic, barium, cadmium, chromium(III), cobalt, copper, lead, mercury, nickel, selenium, tin and vanadate, so we decided described and discussed its role in male infertility. REFERENCES: 1. J.S. Gabrielsen et al. “Chronic exposures and male fertility: the impacts of environment, diet, and drug use on spermatogenesis” Andrology. 4, p. 648-661, 2016. 2. J. Saczko et al. “Estrogen Receptors in Cell Membranes: Regulation and Signaling” Adv Anat Embryol Cell Biol. 227, p. 93-105, 2017. 3. P.D. Darbre. “Metalloestrogens: an emerging class of inorganic xenoestrogens with potential to add to the oestrogenic burden of the human breast” J Appl Toxicol. 26, p. 191-197, 2006. 4. S.V. Rana. “Perspectives in endocrine toxicity of heavy metals – a review” Biol Trace Elem Res.160, p. 1-14, 2014. 5. M. Oettel. “Is there a role for estrogens in the maintenance of men's health?” Aging Male. 5, p. 248-257, 2002.

83 P 30. Pseudocereals as a underestimated source of nutrients U. Kaim1,2, J. Harasym1,2, R. Olędzki1,2, A. Orkusz1,2, L. Bogacz-Radomska1,2

1. Adaptive Food Systems Accelerator – Science Centre, Wroclaw University of Economics, Poland 2. Department of Biotechnology and Food Analysis, Institute of Chemistry and Food Technology, Wroclaw University of Economics, Rising trend for new life style – gluten-free diet, frequently chosen both for medical and non- medical reasons, results in drastic changes in typical Western diet caused by an intention to remove/replace typical wheat/gluten containing food. Unfortunately most of gluten-free products on the market lacks of the nutrients in comparison to their original equivalents. This is caused by manufacturing methods of such – gluten-free products, which explore mainly highly purified ingredients as starches and hydrocolloids [1]. The viable alternative for existing gluten-free products are naturally gluten-free raw materials from which pseudocereals, naturally free of gluten, can be extremely nutritious food. Their chemical content, high level of nutritious fractions, especially fiber and antioxidants characteristics predestinate such plant as human food and wheat substitute [2]. This presentation compares the chemical characteristics of pseudocereals especially buckwheat, amaranth, chia, quinoa and kaniwa. Also summarizes existing knowledge regarding the impact of specific, isolated from pseudocereals bioactive compounds. Pseudocereals are still underestimated in their application in both in agriculture and food manufacturing. Meanwhile, those plants can be found at any latitude and contain lots of micro and macro elements, starch, proteins and fibers [1, 2]. REFERENCES: 1. U. Kaim, J. Harasym, Nauki Inżynierskie i Technologiczne vol.4(27), p. 41-54, 2017. 2. Editor: B. R Hamaker, Woodhead Publishing Limited, 2008.

84 P 31. Recent findings in gluten and wheat related disorders U. Kaim1,2, J. Harasym 1,2, A. Orkusz1,2, R. Olędzki1,2 , L. Bogacz-Radomska1,2

1. Adaptive Food Systems Accelerator – Science Centre, Wrocław University of Economics, Poland 2. Department of Biotechnology and Food Analysis, Institute of Chemistry and Food Technology, Wrocław University of Economics, Cereals and cereal products are a main source of nutrition for 75% world population and can be consumed in different forms, shapes and levels of processing. But in a last few decades the number of patients suffering from disease related with cereal consumption, especially gluten containing products is rising, and most of people with this conditions are undiagnosed and untreated [1, 2]. Gluten is a complex of protein present in the most common consumed cereals such as wheat, barley and rye. It is recognized as main factor contributing to large number of people diagnosed with celiac disease, wheat allergy or suffering for non-celiac gluten sensitivity [3, 4]. However, despite the advanced knowledge about pathophysiology of celiac disease – the factors causing “non celiac gluten sensitivity” are still unclear. The progressing investigations reveal more details of the etiology of gluten/wheat related digestion disorders. It is undisputable that lot of people can observed symptoms triggered soon after gluten ingestion such as abdominal pain, fatigue, joint and muscle pain, depression and others disappearing after few days of gluten free diet [5]. This presentation summarizes the recent findings in specific field of gluten/wheat dependent dysfunctions. Additionally it presents the challenges facing gluten-free market due to rising number of consumers excluding gluten containing products from their diet both for medical and life style reasons. The most important of which is sufficient nutrition level supply as gluten free products available in the market are of poor quality and can contribute to malnutrition to patients already having nutritional deficiencies. REFERENCES: 1. U. Kaim, J. Harasym, Engineering Science and Technologies, vol. 4, p. 41-54, 2017. 2. B. Lebwohl, et al., BMJ 2015;351:h4347. 3. C. Catassi, et al. Nutrients vol. 5, p. 3839-3853, 2013. 4. D. E. Khoury, et al. Nutrients, vol. 10, p. 1-25, 2018. 5. C. Catassi, et al. Nutrients vol. 7, p. 4966-4977, 2015.

85 P 32. The analysis of interaction between Cu(II) ions and cyclopeptides with S-S motif in aspect of biomimetic design M. Kalinowska1, W. Witak1, J. Brasuń1

1Department of Inorganic Chemistry, Wroclaw Medical University, Borowska 211A, 50-552 Wrocław, Poland The cyclopeptides are ubiquitously found in nature. They are a significant substances in living organisms where they play roles as hormones, antibiotics, toxins, molecules with the enzymatic and transporting functions [1, 2]. In recent years scientists have paid particular attention to isolation and study cyclopeptides from natural sources [2]. Many of them possess large therapeutic potential as antibiotics, cytotoxic, antifungal and anticancer agents [3]. The cyclic structure of peptides, that has been obtained by a disulfide bridge or formed by a peptide bond, protects against enzymatic degradation and adverse environmental conditions. Cyclic structure is also characterized by limited mobility of the peptide chain and by higher stability what have impact on the specific binding of divalent metal ions, for example Cu (II). Therefore, cyclopeptides may be compounds that are capable of mimicking the action of natural substances, e.g. enzymes [4, 5]. In this work we present our initial research on coordination abilities between copper (II) ions and cyclopeptides with disulfide bond. The investigated ligand (Scheme 1) consists of nine amino acids. Cyclic structure is obtained here by disulfide bridge between two cysteinyl moieties. In addition, our compound include more than one histidine moiety, what makes it an interesting ligand for the transition metal ions. The cyclopeptide have been studied by potentiometric and spectroscopic methods: UV-Vis and CD. The results allow to characterize the binding properties of Cu (II) complexes formed in different systems. The analysis was carried out in various ligand to metal ratios: 1:1, 1:2, 2:1. In each of these systems, the formation of thermodynamically stable complexes was observed.

Scheme 1. Chemical structure of studied ligand: Ac-c(CHKHPHRHC)-NH2. REFERENCES: 1. Y. Lee, et al. Peptides vol. 95 p. 94-105, 2017. 2. L. Gahan, et al. Polyhedron vol. 153 p. 1-23, 2018. 3. Y. Lee, et al. Peptides vol. 95 p. 94-105, 2017. 4. J.S. Pap, et al. Chem.Commun. vol. 51 p. 6322-6324, 2015. 5. M. Hoarau, et al. Coord.Chem.Rev. vol. 308 p. 445-459, 2016.

86 P 33. Antioxidant potential of fermented tea drinks J. Kałduńska1, J. Janił1, K. Jakubczyk1, K. Janda1, J. Kikut1, W. Żwierełło1, A. Wolska1, D. Styburski 1, M. Skórka-Majewicz1, J. Palma1, N. Komorniak1

1Department of Biochemistry and Human Nutrition, Pomeranian Medical University, Szczecin Kombucha is a fermented tea drink made with the participation of the symbiotic culture of bacteria and yeast (SCOBY). In recent years, the popularity of this product has increased in Poland. Owing to its high content of organic acids, vitamins, amino acids as well as polyphenols, kombucha has a wide spectrum of health-promoting properties. Studies have shown that regular consumption of kombucha may improve the immune system, normalize blood pressure, and regulate intestinal transit. This drink also supports the work of the liver and pancreas, has a positive effect on the mood, adds energy and has a beneficial impact on intestinal bacterial flora. Four types of tea were used for the study: black, green, white and red. The fermentation process was carried out for 14 days. Antioxidative activity was measured spectrophotometrically using the DPPH and the FRAP method. The highest antioxidant potential determined by the DPPH method was observed in beverages prepared using all kinds of tea directly after the addition of the leaven and the SCOBY culture. With the duration of fermentation, the ability to inactivate free radicals decreased. Kombucha prepared from green tea showed the highest values of DPPH inhibition. The highest antioxidant capacity determined by the FRAP method was observed in the green tea kombucha after 1 week of fermentation. The extension of the fermentation period to 14 days resulted in the reduction of the antioxidative potential measured by both the FRAP and DPPH methods. On the basis of the results, it can be concluded that both the type of tea and the time of fermentation of the kombucha affects the amount of antioxidant compounds. It seems reasonable to use high-quality green tea to make a kombucha drink instead of traditionally used black tea.

87 P 34. Neurotoxic effects of fluoride and its impact on cognitive impairment P. Kapczuk1, P. Kupnicka1, E. Metryka1, D. Simińska1, W. Żwierełło2

1Department of Biochemistry, Pomeranian Medical University in Szczecin, Powstańców Wlkp. 72 St, 70-111 Szczecin; Poland 2Department of Biochemistry and Human Nutrition, Pomeranian Medical University in Szczecin, Broniewskiego 24 St, 71-460 Szczecin, Poland In the 1950s in the US, and then in Europe, fluoridation of water began. At that time, it was primarily thought about its anticaries properties. After years of observation, fluoride in excessive amounts turned out to be a toxin for living organisms, that interferes, among others, the functioning of liver, kidneys, thyroid and pituitary gland and brain. The mechanism of fluoride toxicity is not fully understood yet. It is certain that its compounds have the ability to penetrate the placenta, accumulate in the neural tube before birth and may cause developmental disorders in the embryonic period. Studies conducted among newborns born in areas with high concentrations of fluoride in the environment, showed significant changes in their behavior in terms of the response to auditory and visual stimuli in comparison with the control group. In the central nervous system (CNS) fluoride causes the swelling of mitochondria, sticking of chromatin, damage to the nuclear membrane and it affects the synthesis of proteins, thereby delaying the growth of cells and their division. A similar effect may also be exerted by the increased amount of free radicals arising in the CNS caused by the action of fluoride. Under its influence, the number of synaptic connections is reduced, what may have a devastating effect on the efficiency of cognitive processes. In epidemiological studies, it has been proved that high concentration of fluoride in drinking water causes decreased IQ levels in children, negatively affects cognitive development and lowers the ability to learn and remember. Similar observations have been made in industrial workers who were exposed to inhalation of fluoride compounds. The deterioration of memory and psychomotor performance has also been observed. Some studies suggest a relationship between the occurrence of ADHD among children and adults, and high concentrations of fluoride in drinking water. In animal studies, it has been proved that fluoride disrupts the synthesis of neurotransmitters (including epinephrine, serotonin, dopamine, glutamate) and their receptors, leading to the development of neurodegenerative changes in the CNS. Changes in the concentration of neurotransmitters are characteristic of degenerative diseases of the nervous system and they are also observed in mental diseases. Fluoride can therefore play a role in the formation and development of these disorders. Studies carried out so far prove that fluoride may have a negative impact on development and may contribute to the formation of diseases associated with the nervous system, however, the exact knowledge of its toxicity mechanisms requires additional research. REFERENCES: 1. Choi AL, Sun G, Zhang Y, Grandjean P. “Developmental Fluoride Neurotoxicity: A Systematic Review and meta-analysis” Environmental Health Perspectives, 2012; 10 (120) 1362-1368. 2. Malin AJ, Till C. “Exposure to fluoridated water and attention deficit hyperactivity disorder prevalence among children and adolescents in the United States: an ecological association” Environ Health. 2015 Feb 27;14:17. 3. Pereira M, Dombrowski P, Losso E, Chioca L, Da Cunha C, Andreatini R. “Memory Impairment Induced by Sodium Fluoride is Associated with Changes in Brain Monoamine Levels” Neurotoxicity Research,2011; 55-62. 4. Valdez Jiméneza L et al. “In utero exposure to fluoride and cognitive development delay in infants” Neurotoxicology. 2017 Mar;59:65-70.

88 P 35. The problem of drug dosing in obese children A. Kawalec

Department of Hygiene, Wroclaw Medical University, Wrocław, Poland Recently an increase in the prevalence of childhood overweight and obesity has been observed. According to World Health Organization 1 in 5 children and adolescents are overweight or obese. It is estimated that in Poland the problem concerns 16.4% of paediatric population. Obese children are at risk for many health consequences, and therefore are more likely to receive drug therapy in comparison to non-obese children. Obesity is accompanied by changes in body composition, such as different proportion of both lean body mass and fat mass, and also pathophysiological alterations. These may affect differences in pharmacokinetics and pharmacodynamics, and result in under- or overdosing if traditional paediatric dosing strategies are used. There have been multiple size descriptors proposed for the scaling of the dose of a drug in children, including total body weight and body surface area. However, the doses of drugs used in obese patients are largely based on empiricism and experience of prescribers. Pharmacokinetic variables often used to determine the dosage of a medication are volume of distribution and clearance. In obese children volume of distribution is best represented by total body weight, and clearance by lean body weight. In addition, the relative degree of drug lipophilicity and hydrophilicity should also be taken into consideration. In scope of limited data about drug dosing in obese children, there is a need for more studies to ensure efficacy of pharmacotherapy and enhance drug safety in this group of paediatric patients.

89 P 36. Strategies for immune therapy in Melanoma A. Kiełbik1, S. Kwiatkowski1, D. Przystupski1, P. Wawryka1, K. Kotowski1, J. Saczko2, J. Kulbacka2

1Faculty of Medicine, Wroclaw Medical University, J. Mikulicza-Radeckiego 5, 50-345 Wroclaw, Poland 2Department of Molecular and Cellular Biology, Wroclaw Medical University, Borowska 211A, 50-556, Wroclaw, Poland Though years of studies for cancer biology and immunology, melanoma, in it’s late stage, still remains lethal for patients and cause a decent problem to physicians. Melanoma of skin is estimated to be responsible for 0.6% of deaths caused by tumor disease. With over 300,000 new cases and 60,000 deaths estimated globally, melanoma is one of the most frequently diagnosed cancer in North America, Australia and New Zealand [1]. Large diversity, rapid grow, and neglection of early signs and symptoms often leads to late diagnosis [2]. Once melanoma has been detected in its early stage, it can be cured by surgical resection, but with further progression to metastatic stage it is extremely difficult to treat it. Long studies of cell–signaling and mechanisms of immune system have contributed to development of new therapeutic approaches [3]. Although, the clinical application of immune therapeutics provided satisfactory results, significantly extended the survival of patients, in some cases the response to treatment wasn’t so evident. As a consequence of that, the most present research explore the mechanisms of resistance to immune treatment such us, avoiding detection via MHC I down regulation, production of inhibitory microenvironment and accumulation of immunosuppressive lymphocytes and myeloid cells. The future task is to determine which of these mechanisms are important in general and which decide about the immune status of individual patients [4]. This review brings into discussion the mechanism of immune response, the most present possibilities of immunotherapy together with resistance strategies of melanoma cancer cells. ACKNOWLEDGEMENTS The research was supported by the Scientific Cancer Cell Biology Group No. 148 (WMU) and by “Najlepsi z Najlepszych 3.0” program of Polish Ministry of Science and Higher Education REFERENCES: 1. F. Bray, F. Jacques, S. Isabelle, L. S. Rebecca, A. T. Lindsey, and Jemal Ahmedin, “Global Cancer Statistics 2018: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries,” CA CANCER J CLIN, vol. 68, no. 68, pp. 394-424, 2018. 2. P. F. Hanrahan, P. Hersey, S. W. Menzies, A. B. Watson, C. A. D. Este, and R. P. Alfred, “Examination of the Ability of People to Identify Early Changes of Melanoma in Computer-Altered Pigmented Skin Lesions,” ARCH DERMATOL, 1997. 3. V. Gray-Schopfer, C. Wellbrock, and R. Marais, “Melanoma biology and new targeted therapy,” Nature, vol. 445, no. 7130, pp. 851-857, 2007. 4. I. Nisbet, “Cancer immunotherapy comes of age (Finally!),” Australas. Biotechnol., vol. 26, no. 2, pp. 38-40, 2016.

90 P 37. In vitro study on the effect of blue light LED-based photodynamic therapy on glioblastoma multiforme A. Kiełbik1, P. Wawryka1, D. Przystupski1, S. Kwiatkowski1, K. Kotowski1, J. Saczko2, J. Kulbacka2 1 Faculty of Medicine, Wroclaw Medical University, J. Mikulicza-Radeckiego 5, 50-345 Wroclaw, Poland 2Department of Molecular and Cellular Biology, Wroclaw Medical University, Borowska 211A, 50-556, Wroclaw, Poland Glioblastoma multiforme is also known as grade 4 glioma due to the fact that it is the most aggressive among this histological type of cancers. Unfortunately, not only it is characterized by poor prognosis for patients but also the highest prevalence comparing with other gliomas [1]. In our in vitro research we used curcumin (CUR) – diferuloylmethane derived from the Indian spice, turmeric – since CUR has been reported to have apoptotic and antiproliferative activity against multiple types of cancers [2]. To enhance the cytotoxic effect of CUR we used its photosensitizing properties in photodynamic therapy (PDT) [3]. The aim of this research was to define the optimal concentration of curcumin for usage in photodynamic therapy that would induce cytotoxic effect on glioblastoma cells and would not affect healthy human cells. In the study we cultured human glioblastoma multiforme cell line SNB19. The cells were grown under the standard, sterile culture conditions in sealed polystyrene flask. Consequently, they were harvest and bred in the environment of different curcumin concentrations. The photodynamic protocol was carried out after 2h, 24h and 48h. To measure the cell viability by the mitochondrial activity MTT cytotoxicity assay was performed on every sample both with and without PDT. The results show that in the ambience of curcumin glioma cells have lower viability. The addition of PDT and usage of curcumin as a photosensitizer increased cytotoxic effect on the cancer cells reaching respectively for times 2h, 24h and 48h - IC50=24.91μM, EC50=21.57μM and EC50=28.07μM curcumin concentration. Curcumin-based treatment is promising medicament and photosensitizer for human glioma treatment. The PDT gives selectivity and enhance the treatment. Further investigation on human glioblastoma multiforme cells and normal human cells is required to fully understand direct cellular effect and side effects of curcumin-based PDT therapy. ACKNOWLEDGEMENTS The research was supported by the Scientific Cancer Cell Biology Group No. 148 (WMU) and by “Najlepsi z Najlepszych 3.0” program of Polish Ministry of Science and Higher Education REFERENCES: 1. Holland, Eric C. “Glioblastoma multiforme: the terminator” Proceedings of the National Academy of Sciences 97.12 (2000): 6242-6244. 2. Anand, Preetha, et al. “Curcumin and cancer: an “old-age” disease with an “age-old” solution” Cancer letters 267.1 (2008): 133-164 3. Koon, H. K., et al. “Photodynamic effect of curcumin on NPC/CNE2 cells” Journal of environmental pathology, toxicology and oncology 25.1-2 (2006).

91 P 38. The effect of high-fat and high-cholesterol diet on the level of short-chain fatty acids J. Kikut, N. Komorniak,W. Żwierełło, J. Kałduńska, A. Wolska, D. Maciejewska, J. Palma, D. Styburski, M. Skórka-Majewicz, M. Popik, D. Matyniak, A. Fryda, E. Stachowska Department of Biochemistry and Human Nutrition, Pomeranian Medical University, Szczecin, Poland Introduction: Diet has a significant impact on the intestinal microflora composition. The level of short-chain fatty acids (SCFA) is strongly associated with dietary fiber intake. SCFAs are produced by the fermentation of undigested carbohydrates and proteins in the large intestine by anaerobic microflora. Recent studies have shown than Western diet, rich in sugar and saturated fats, can cause dysbiosis. Aim: The aim of the study was to evaluate the effect of high-fat, high-cholesterol diet (HFHCh) on the ratio of short-chain fatty acids. Material and methods: 30 Male Sprague–Dawley rats were fed a high fat diet (HFD) consisting of 88 g of a normal diet, 10 g of lard oil, and 2 g of cholesterol. Control rats were fed a normal diet. The rats were killed 2, 4, 8, 12, 16 and 20 weeks after HFD exposure. During every section, rat feces were collected and SCFA were analyzed using gas chromatography. Results: Rats fed with HFHCh showed a significant decrease in butyric acid and significant increase in propionic acid concentration compared to the control group. In the control group, the main fatty acid was acetic acid and propionic acid. Conclusions: High-fat and high-cholesterol diet changes the ratio between individual fatty acids. It seems that the proper ratio between individual acids has an impact on the prevention of metabolic diseases. Changes in the proportion of SCFA in the gut may affect the integrity and function of the intestinal barrier, which can lead to metabolic disorders.

92 P 39. HPMC based High Internal Phase Emulsion as a route to novel porous polymer scaffolds J. Kłak, M. Nowak, K. P. Nartowski, B. Karolewicz

Department of Drug Form Technology, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, Wroclaw, Poland Introduction: Porous polymers with well-defined porosity and high specific surface area are being used in a wide range of applications such as tissue engineering, three-dimensional cell cultures, catalysis, water retention, shape memory materials or as templates for the production of porous inorganics and porous carbons. polyHIPEs (porous emulsion-templated polymers synthesized using high internal phase emulsions method – HIPE) are currently of great scientific and technological interest as they provide the advantage of designing highly porous well-defined matrices in a tailored manner [1,2]. In this study hydroxypropyl methylcellulose (HPMC) was used as a main component of the scaffolds. Hypromellose is widely used as a food additive (an emulsifier, thickening or suspending agent). Furthermore, it is biodegradable, biocompatible, viscoelastic polymer used in eye drops, as well as an excipient and controlled-delivery component in oral medications. Experimental: Synthesis of HPMC porous scaffolds were performed as follows. In the first step 3 g of HPMC was dissolved in 60 ml of DMF to obtain clear colorless solution and functionalized using 1, 1.5, or 2 g of methacrylic anhydride (ME). Methacrylated HPMC (M-HPMC) was than purified by 5 days dialysis and lyophilized. Functionalization of HPMC was confirmed by FTIR and 1H and 13C NMR spectroscopy. In the next step HIPEs were prepared by dissolving M-HPMC in water with potassium persulfate (cross-linking initiator) and subsequently dispersing phase (tetradecane, ethyl acetate, n-hexane and castor oil) was added dropwise during continuous vortexing. Obtained emulsions were polymerized in oven at 60°C by 48 h and yielded porous polymer were purified in Soxhlet apparatus using ethanol and water and lyophilized. Structure of scaffolds were confirmed by optical and electron microscopy. Results: It was found that to obtain M-HPMC with preserve high water solubility, an optimal HPMC:ME mass ratio in functionalization step is 3:1. All prepared HIPEs were stable during polymerization process. Microscopic investigation revealed that pore size depends from type of used dispersed phase and smallest pores with diameter bellow 50 μm were obtained using ethyl acetate. HPMC based porous scaffolds due to their biocompatibility may be considered as a promising material for tissue engineering. REFERENCES: 1. Kimmins, S. D. & Cameron, N. R., Adv. Funct. Mater. 21, 211-225 (2011). 2. Silverstein, M. S., Polymer (Guildf). 55, 304-320 (2014).

93 P 40. Intermolecular interactions of newly designed nutulin-3 derivatives and MDM2 protein: computational approach T. Kłosiński1, A. Stasik1, E. Kiełbasa1, D. Gula1, Ż. Czyżnikowska2

1Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, 50-556 Wrocław, Poland 2Department of Inorganic Chemistry, Wroclaw Medical University, 50-556 Wrocław, Poland Tumor suppressor p53 protein plays an important role in many signaling pathways. For example it maintains genomic stability by activation of DNA-repair mechanism or inducing apoptosis in cells of living organisms. The most common mechanism of p53 inhibition is interaction with MDM2 protein which has significant role in above half of all human tumor cells [1]. Additionally, MDM2 is responsible for its ubiquitination and proteasomal degradation. Moreover, the overexpression of mentioned oncoprotein promotes tumorigenesis [2]. Therefore, many potent and selective inhibitors of the p53–MDM2 interaction were lately design as a potential anticancer agents [3]. It should be mentioned that the basis of p53 and MDM2 interactions are not fully understood. Crystallographic data revealed that Leu26, Trp23 and Phe19 motif in tumor suppressor protein is key sequence responsible for p53-MDM2 complex creation. Therefore, the attention focuses on discovering of molecules that can mimic these p53 residues [4]. Nutulin-3 is the small molecule which can bind to the binding pocket of MDM2 and inhibit protein-protein interaction what can induce a growth-inhibiting state in cancer cells. In the present study the intermolecular interactions of three newly designed nutulin-3 derivatives with MDM2 protein were characterized using molecular docking simulation. We predict the binding mode of analyzed compounds within the active site of protein. The obtained binding energies and inhibition constant for analyzed system allow to consider them as a promising MDM2 inhibitors. REFERENCES: 1. A. Gupta, et al. Biomedicine & Pharmacotherapy vol. 109 p. 484-492, 2019. 2. G. Popowicz et al. Angew Chem int Ed engl. vol. 50(12) p. 2680-2688, 2011. 3. S. Shangary et. al. Annu Rev Pharmacol Toxicol. vol. 49 p. 223-241, 2009. 4. P. H. Kussie, et al .Science vol. 274 p. 948-953, 1996.

94 P 41. Chemical probes to detect Granzyme A activity S. Kołt1, T. Janiszewski1, S. Snipas2, D. Kaisermann3, P. Bird3, N. Bovenschen4, M. Drąg1, G. Salvesen2, P. Kasperkiewicz1

1 Wroclaw University of Science and Technology, Wroclaw, Poland, 2 Sanford Burnham Prebys Medical Discovery Institute, La Jolla, USA 3 Monash University, Clayton, Australia 4 UMC Utrecht, Utrecht, Netherlands Granzymes (granule enzymes) are members of the serine protease family and are widely present within cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. These proteases, located in cytolytic granules, are released after contact with a target cell, in which they activate distinct cell death pathways, displaying a crucial role in protecting hosts from viral infection, cancer and cell transformation. There are 5 granzymes identified in humans: GrA, GrK, GrB, GrH and GrM, of which GrA is the most abundant serine protease in killer cell granules. Granzyme A activates a distinct cell death pathway that does not include caspases, yet it is morphologically indistinguishable from apoptosis. Upon entrance into a target cell, this serine protease leads to DNA damage by disrupting mitochondrial metabolism and generating reactive oxygen species. Except for its cell death inducing function, granzyme A is also known to be involved in regulation of B-cell proliferation and can act as a T and NK cells migration mediator. Among all granzymes, GrB has been intensively studied for many years, in contrast, there is very limited information about the localization and function for the remaining granzymes. This may be due to the fact, that there are no reliable tools for granzyme investigation. Antibodies, commonly used in proteases research, do not distinguish between active and inactive forms of the enzymes. Therefore, we focused on finding an optimal technique for granzyme investigation. As a proof of concept, we used granzyme A and found a lead peptide-based sequence, which was transformed into an irreversible probe and labels active granzyme A. This probe will be used to reveal granzyme A functions, involving both lysates and whole cells assays. ACKNOWLEDGEMENTS This work was supported by HOMING Programme, a Grant Project of the Foundation for Polish Science, funded by the European Union under agreement No. 2016-3/24. PK is supported by Ministry of Science and Higher Education in Poland.

95 P 42. Effect of disease activity on parameters of oxidative-antioxidant balance in rheumatoid arthritis patients I. Kokot1, A. Piwowar2, E. M. Kratz1, S. Płaczkowska3, K. Sołkiewicz1, L. Pawlik-Sobecka1, R. Sokolik4 1Department of Laboratory Diagnostics, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, Wroclaw, Poland 2Department of Toxicology, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, Wroclaw, Poland 3Diagnostics Laboratory for Teaching and Research, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, Poland 4Department and Clinic of Rheumatology and Internal Medicine, Faculty of Medicine, Wroclaw Medical University, Poland Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with joint destruction. Many data suggest an important role of oxidative stress in the pathogenesis and severity of RA . The aim of this study was to assess the correlations between parameters of oxidative-antioxidant balance and the disease activity in patients with RA. RA activity in patients was calculated as disease activity score 28 (DAS28), according to 28 swollen and tender joints with erythrocyte sedimentation rate (ESR) and visual analog score (VAS). The study group consisted of 33 RA patients (49±16 age), including 16 with low disease activity (DAS28≤3.2) and 17 with moderate and active disease (DAS28>3.2). The control group consisted of 18 healthy subjects. In the blood serum samples the total antioxidant status (TAS) was measured with the commercial kit (Randox, UK) and total oxidant status (TOS) based on the method developed by Erel was determined. Patients with low disease activity had significantly higher total antioxidant status and significantly lower total oxidant status in comparison to RA patients with moderate and active disease. We also observed a negative correlation between TAS and DAS28 and positive correlation between TOS and DAS28 in RA patients. This study indicates that the parameters determining the oxidative-antioxidant balance may be used as the additional markers of disease activity in patients with RA. It suggest that improving the total antioxidant status should be reflected in a reduction in disease activity. ACKNOWLEDGEMENTS This study was funded by a statutory grant awarded by the Ministry of Science and Higher Education, grant number: ST-960. REFERENCES: 1. M. Veselinovic , N. Barudzic , M. Vuletic, V. Zivkovic , A. Tomic-Lucic, D. Djuric , V. Jakovljevic “Oxidative stress in rheumatoid arthritis patients: relationship to diseases activity” Mol Cell Biochem.; 391(1-2):225-32, 2014. doi: 10.1007/s11010-014-2006-6. 2. O. Erel “A new automated colorimetric method for measuring total oxidant status” Clinical Biochemistry 38, 1103-1111, 2005. 3. H.M. Khojah, S. Ahmed, M.S. Abdel-Rahman, A.B. Hamza “Reactive oxygen and nitrogen species in patients with rheumatoid arthritis as potential biomarkers for disease activity and the role of antioxidants” Free Radic Biol Med., 97, 285-291, 2016. doi: 10.1016/j.freeradbiomed.2016.06.020. 4. A.R. Phull, B. Nasir, I.U. Haq, S.J. Kim “Oxidative stress, consequences and ROS mediated cellular signaling in rheumatoid arthritis” Chem Biol Interact. 1(281), 121-136, 2018. doi: 10.1016/j.cbi.2017.12.024.

96 P 43. Vitamin D status among burn patients N. Komorniak1, J. Palma1, J. Kikut1, J. Kałduńska1, A. Wolska1, M. Skórka-Majewicz1, W. Żwirełło1, D. Styburski1, A. Krajewski2, K. Piorun2, D. Maciejewska1, M. Markowska2, E. Metryka3, K. Skonieczna-Żydecka1, E. Stachowska1

1Department of Biochemistry and Human Nutrition, Pomeranian Medical University in Szczecin, Poland 2West Pomeranian Center of Treating Severe Burns and Plastic Surgery in Gryfice, Gryfice, Poland 3Department of Biochemistry, Pomeranian Medical University in Szczecin, Poland Introduction. Burns are one of the most serious injuries, which often includes multi-organ dysfunction. Every year, about 1% of Polish people (both children and adults) suffer from various types of burns. Aim. The aim of the study was to evaluate the vitamin D concentration according to burn degree. Material and Methods. 126 patients with burns injuries were enrolled in the study. Patients were qualified due to thermal burns - over 10% of the body surface area (BSA). The vitamin D concentration was measured in commercial certified laboratory in the Hospital. The statistical analysis was performed using the “R 3.0.2” program. In the order to estimate connection between burn degree and concentration of vitamin D Poisson regression was used. The values being at the threshold of statistical tendency were established from p<0.05 were considered as statistically important. Results. Almost all patients (92%) in study group had an improper level of vitamin D (<30 ng/ml), with the average of 11.6 ng/ml ± 10.7 ng/ml. 17.5 % of patients had level below the limit of determination - under 3 ng/ml. Only 8% of study group had a proper concentration of vitamin D (>30 ng/ml). Poisson regression showed that there is statistical tendency toward the associated between vitamin D concentration and burn degree. Conclusion. Thermal injury influence vitamin D levels. Vitamin D is frequently found to be deficient and may potentially influence short- and long- term outcomes in burn patients.

97 P 44. From physiology to pathology: the phenomenon of cellular senescence M. Kotas1, M. Tyliszczak1, P. Zuziak1 1Faculty of Medicine, Wroclaw Medical University, Wroclaw, Poland Cellular senescence was first discovered by Hayflick and Moorhead over fifty years ago when they noticed that their fibroblast colonies ceased to divide after approximately 50 divisions. Nowadays we know that senescence comes in many shapes and sizes, and more recent studies suggest that it may play an important role in tissue remodelling by recruiting immune cells through senescence-associated secretory phenotype (SASP), thus helping to clear damaged or otherwise malfunctioning cells. Furthermore, it seems to be a part of normal embryonic development. When not working properly, however, it can lead to accumulation of tissue damage and chronic inflammation, which may in turn cause various pathologies. In our poster, we would like to show the role cellular senescence plays in human physiology and take a quick look at the possibilities of applying this knowledge in a clinical setting, particularly in cancer therapy.

98 P 45. Can glycation be an epigenetic modification? A. Kotowska1, K. Rajtczak-Wielgomas1, D. Indyk2, K. Nowakowska3 A. Kuzan2

1Department of Histology and Embryology, Wroclaw Medical University, Wrocław, Poland 2 Department of Medical Biochemistry, Wroclaw Medical University, Wrocław, Poland 3 Department and Clinic of Internal Medicine and Allergology, Wroclaw Medical University, Wrocław, Poland Currently, it is assumed that epigenetic modifications of histones include acetylation, methylation (mono-, di- or tri-molecular), phosphorylation, ubiquitination and conjugation with SUMO molecules (small ubiquitin-likemodifier), so-called sumoylation. Since histones are proteins with a very high content of basic amino acids and are proteins with a relatively long half-life (4-5 months), we postulate that the protein may be susceptible to glycation. The question is whether glycation can be an epigenetic modification? In order to verify the hypothesis, as a preliminary study it was planned to check whether there is a colocalization of histones and AGE products. A number of immunoenzymatic reactions on human aortic section specimens was carried out. Reactions with phalloidin and LipidTOXTM were also tested. Advanced glycation end products (AGE) have been shown to be present in the inner layer of the artery as well as in the muscular and adventitia layer. In advanced plaque, where there are cholesterol fissures, fat cores and / or calcification areas, the AGE antigen is spotted, but it is also unrelated to nuclear proteins, because the reaction with DAPI and anti- histone antibodies indicates a lack of viable cells in such atherosclerotic regions of the tissue. Single samples showed co-localization of histone H1 and H2A with AGE. The results suggest that histone glycation in the arteries is possible, but it is not a common or intense phenomenon. The inclusion of glycation in group of epigenetic modifications would open new perspectives for the diagnosis and treatment of atherosclerotic diseases, therefore continuation of research seems necessary and valuable.

The work was financed by the National Science Center (decision number DEC 2017/01 / X / NZ9 / 00599).

99 P 46. Advanced glycation end products (AGEs) and receptors for advanced glycation end products (RAGE) in pathologies of human thyroid gland – a pilot study Ł. Kotyra

Department of Medical Biochemistry, Wroclaw Medical University, Wroclaw, Poland Background: Advanced glycation end products (AGEs) are chemical compounds formed in a process of glycation. Glycation is a non-enzymatic reaction between free amino groups of proteins and free carbonyl groups of reducing sugar [1]. Glycative stress may cause an initiation and progression of cancer in different organs including human thyroid gland [2]. Aim: The aim of the study was to investigate if advanced glycation end products (AGEs) and their receptor (RAGE) are present in different pathologies of thyroid gland compared to healthy tissue. The relationship between the amount and localization of these antigens was also sought. Materials and methods: In our experiment 30 samples of thyroid gland (13 from control healthy area, 7 pathological and 10 from fatty tissue) were subject to analysis by immunohistochemistry for presence of AGE and 7 specimen (3 pathological, 2 healthy and 2 from fatty tissue) were analysed for presence of RAGE. Results: The intensity of immunohistochemistry reaction differed in individual examined samples. It was the most intense in the pathological samples of thyroid gland. The AGE antigen was present in connective tissue in all specimen of healthy gland and tumor. The receptor RAGE occurs in about half of the cases, but no pattern has yet been found Conclusions: AGEs are present not only in oncological samples but also in inflammation, goiter and healthy tissue of thyroid gland. Research into the presence of RAGE receptors in the greater number of samples should be continued to determine patterns in their presence and content in thyroid tissue. REFERENCES: 1. Ahmad S, et al. AGEs, RAGEs and s-RAGE; friend or foe for cancer. Semin Cancer Biol., Apr;49: 44-55,2018. 2. Ahmad S, et al. Do all roads lead to the Rome? The glycation perspective! Semin Cancer Biol., Apr;49:9-19,2018.

100 P 47. OTC Herbal Products and Dietary Supplements Using by Patients Receiving Chemotherapy – Survey Study A. Kowalczyk1, A. Bodalska1, K. Boszkiewicz2 , K. Karłowicz-Bodalska3

1Department of Pharmacognosy and Herbal Medicines, Wroclaw Medical University, Faculty of Pharmacy with Division of Laboratory Diagnostics Wroclaw, Poland 2HASCO-LEK, Pharmaceutical Company, Wroclaw, Poland 3Department of Industrial Pharmacy, Wroclaw Medical University, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw, Poland People with cancer often consider taking supplements and herbal products [1]. Many hope that these products improve health, provide nutrition, boost the immune system or reduce side effects of the pharmacotherapy [2]. Among cancer patients receiving chemotherapy, there is a growing trend to use self-medication with preparations sold over the counter (OTC) and dietary supplements containing plant raw materials [3]. The aim of the study was to evaluate some aspects of the herbal OTC drug and dietary supplement usage among patients treated on the chemotherapy ward of the Lower Silesian Oncology Center in Wroclaw (Poland). An anonymous survey was conducted among 92 patients with the approval of the Bioethical Commission of the Medical University in Wroclaw (No.: KB-562/2014) and The Declaration of Helsinki in January 2015. The statistical analysis was developed using Microsoft Office Excel 2007 and STATISTICA 10 (StatSoft Inc.) programs. Analysis was performed by χ2 test. The study has confirmed that the use of herbal OTC products and dietary supplements among chemotherapy patients is a common occurrence. The Internet is the main source of information about herbal preparations and the most common place of purchase is a pharmacy [4]. It has been shown that among products of plant origin, there are those which can cause interactions with co- administered synthetic drugs [5]. In conclusion, one can say that it is necessary to educate patients receiving chemotherapy to increase their awareness of the importance of a doctor’s knowledge about all preparations that they take and pharmacists play a very important role in self-medication of the patients. REFERENCES: 1. S.M. Alsanad et al. “An assessment of the impact of herb-drug combinations used by cancer patients” BMC. Complement Altern Med. 16:393, 2016. 2. M.S. Ali-Shtayeh et al. “Complementary and alternative medicine use among cancer patients in Palestine with special reference to safety-related concerns” J Ethnopharmacol. 187:104-22, 2016. 3. L. Butch. “Supportive Care for Cancer Patients During and After Chemotherapy Treatment” JCM. 116: 11-23, 2018.. 4. P. Bastani et al. “Community pharmacy-based survey on pharmacists’ knowledge, attitude, and performance regarding dietary supplements: evidence from south of Iran” J Physiol Pharm Pharmacol. 7:396-402, 2017. 5. E. Ben-Arye et al. “Potential risk associated with traditional herbal medicine use in cancer care: a study of Middle Eastern oncology health care professionals” Cancer. 122:598-610, 2016.

101 P 48. Impact of marijuana consumption on human acute pancreatitis K. Kowalska1, M. Ściskalska2, H. Milnerowicz2

1 Student research group of Department of Biomedical and Environmental Analyses, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, Poland 2Department of Biomedical and Environmental Analyses, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, Poland Background: For centuries people have been using dried cannabis sativa flowers (marijuana) for recreational purposes. However, it has been demonstrated smoking marijuana is a cause of many ailments, such as depression, insomnia and anxiety [1]. The culprit of these effects is most likely THC contained in the plant. The efforts to explain some of the abnormal processes triggered by marijuana consumption have not been entirely successful to this day [1, 2]. Aims: Analysis of sources describing impact of marijuana usage on presence of pancreatic diseases, specifically acute pancreatitis. Overview: In scientific literature there are described various cases of patients suffering from acute pancreatitis, for whom the most common causes of this ailment, such as: excessive use of alcohol, genetic predispositions, infections, gallbladder and gall tract diseases – were debarred [4]. The common factor of these cases is addictive usage of significant quantities of cannabinoid-containing psychoactive substances by the patients before experiencing the symptoms. During physical examination the patients showed symptoms typical for acute pancreatitis. Utilization of morphology and biochemistry blood tests, as well as USG, proved this initial diagnosis to be correct. As a result of implementing symptomatic treatment and preventing patients from using cannabinoids, acute pancreatitis has receded. No relapses have been noted. It was suggest that cause of the disease was activation of CB receptors by cannabinoids (e.g. Δ9-THC), which in turn prompted overproduction and premature activation of pancreatic enzymes [3, 4]. Conclusions: Excessive intake of marijuana is a relevant factor, that may result in developing acute pancreatitis. Presented cases aim to bring closer attention to health issues that marijuana usage brings. REFERENCES: 1. Pharmacokinetics and Pharmacodynamics of Cannabinoids F. Grotenhermen, Nova-Institut, Hürth, Germany, Clin Pharmacokinet 2003; 42 (4). 2. Presence of functional cannabinoid receptors in human endocrine pancreas, F. J. Bermúdez-Silva & J. Suárez & E. Baixeras & N. Cobo & D. Bautista & A. L. Cuesta-Muñoz & E. Fuentes & P. Juan-Pico & M. J. Castro & G. Milman & R. Mechoulam & A. Nadal & F. Rodríguez de Fonseca, Diabetologia (2008) 51:476-487. 3. Case Report: Cannabis-induced acute pancreatitis, American Journal of Emergency Medicine (2011) 29 4. Clinical profile of cannabis-associated acute pancreatitis, Digestive and Liver Disease 49 (2017) 1284-1287.

102 P 49. A new perspective on eye medicines application – colour-changing contact lenses P. Kowalska1 , B. Kupaj1, K. Marciniak1, I. Raudner1, E. Sawicka2

1Students Scientific Society at the Department of Toxicology, Wroclaw Medical University, Poland 2Department of Toxicology, Medical University in Wroclaw, Poland Introduction: According to data from 2015, 82% of the population after 50 years of age suffers from vision disorders: cataract, glaucoma, macular degeneration, diabetic and hypertensive retinopathy, dry eye syndrome and others. Most ophthalmic drugs are available in the form of eye drops or eye ointments. It is difficult to say what dose actually reaches the eye. Moving eyelids and rinse out tears, allow to avoid infection and mechanical damage to the eye by foreign objects, but at the same time they reduce the absorption of the active substance. Methods: After applying the drops to the conjunctival sac, the drug mixes with the tears and stays there for about 2-5 minutes, and then it is removed through the blood vessels of the conjunctiva and through the tear ducts. Only 1-7% of the substance administered in this way reaches the target tissues. Results: Contact lenses could become the solution, changing its colour gradually during the drug releasing process. Thanks to them, doctors and patients can easily evaluate if the drug is delivered to the right place. Lenses are made by using molecular imprinting - a technique that creates molecular cavities in the polymer structure, matching the size and shape of a molecule of a particular compound, such as a drug. During laboratory tests, the contact lenses were saturated with timolol, used in glaucoma treatment. After that they were rinsed in a solution of artificial tears. When the drug have been released, the arrangement of the nearby material particles has changed, and as a consequence – also the color of the lenses was changed. The color change (from green to blue) was visible with the naked eye. Conclusion: This innovative form of drug concentration control give hope for the improvement of the treatment of eyes diseases.

103 P 50. Protective role of testosterone in the development of asthma P. Kowalska1 , B. Kupaj1, K. Marciniak1, I. Raudner1, E. Sawicka2

1Students Scientific Society at the Department of Toxicology, Wroclaw Medical University, Poland 2Department of Toxicology, Medical University in Wroclaw, Poland Bronchial asthma affects about 300 million people around the world. Incidence and severity of the disease are greater in women than in men. The pathogenesis of asthma is multifactorial, recent studies show that the level of sex hormones plays a significant role in the development of asthma. There are differences in incidence depending on gender: considering children, boys are more likely to suffer than girls, but this tendency is reversed among adults. ILC2 (type 2 innate lymphoid cells) play a significant role in the pathogenesis of asthma. Inflammatory proteins – cytokines produced by ILC2 cause inflammation and stimulate the production of mucus in the lungs, which makes breathing difficult. During studies in patients with asthma, greater amounts of ILC2 were found among asthmatics than in healthy people. In the female population, a higher level of ILC2 was observed compared with men, but this difference did not occur in the healthy population. The aim of the research was to determine if the level of ILC2 may be dependent on sex hormones. For this purpose, mouse ILC2 cell cultures isolated from the lungs of adult mice of both sexes were used. In contrast to testosteron, which inhibited cytokine production when added to the coulture, estrogen or progesteron did not cause any change. The discovery of the sensitivity of ILC2 to testosterone can be used to develop targeted drug therapies in allergic diseases.

104 P 51. Molecular docking and QSAR study of newly designed compounds as selective sirtuin II inhibitors A. Krajnik1, Ż. Czyżnikowska2

1Faculty of Pharmacy, Wroclaw Medical University, Borowska 211a, 50-556, Wroclaw, Poland 2 Department of Inorganic Chemistry, Faculty of Pharmacy, Wroclaw Medical University, Borowska 211a, 50-556, Wroclaw, Poland Sirtuins are a family of NAD+ dependent deacetylase proteins that carry out the removal of the acetyl group from various substrates [1, 2]. They fulfill the regulatory function and participate in the differentiation of myocytes and adipocytes. They can also determine the biological life expectancy, including mitosis and apoptosis, gluconeogenesis, neurodegeneration or insulin secretion [3, 4]. It has also been shown in many studies, that sirtuins are likely to play significant role in progress of cancer or Parkinson's disease. Therefore this group of proteins is suggested as potential molecular target in the design process of new drugs [5, 6]. In the present study ten N-(3-(phenoxymethyl)phenyl)acetamide derivatives were considered as a future selective inhibitors of situin II. Their biological properties and interactions with binding site of protein were investigated based on the QSAR analysis and molecular modeling symulations. REFERENCES: 1. B. Karaman et al. European Journal of Medicinal Chemistry, Elsevier Masson SAS vol. 93 p. 584-598, 2015. 2. M. Schiedel et al. Angewandte Chemie vol. 55 p.2252-2256, 2016. 3. Y. Keng et al. Archiv der Pharmazie Chem. Life Sci., vol. 349 p. 1-8, 2016. 4. S. Sakkiah et al. PLoS ONE vol.8 p.1-8, 2013. 5. J. Hu et al. Future Med Chem. vol. 6 p.945-966, 2014. 6. L. Yang et al. Science China Life Science vol. 60 p. 249-256, 2017.

105 P 52. Biotechnological impact of carotenoids produced by halophilic archaea K. Krawczyk, Z. Juszczyk, E. Okła, M. Kowalewicz-Kulbat

Department of Immunology and Infectious Biology, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland

Carotenoids are the group of naturally occurring pigments, mainly lipophilic isoprenoids (C40) with colour range from colourless to yellow, orange and red. Carotenoids are used inter alia as a colorants (food and cosmetics), feed additives for poultry, livestock, fish, and crustaceans or enhancers of antibody in vitro production. In live organisms carotenoids can be precursors for vitamin A synthesis and retinoid compounds required for morphogenesis and embryonic development. Moreover, carotenoids are known as antioxidants, which can supress harmful effect of reactive oxygen species (ROS) and nitrogen oxidative species (NOS). In literature, plants, algae, yeast, cyanobacteria and fungi have been described as a good source of carotenoids. However, only few studies analysed the role of halophilic microorganisms, especially halophilic archaea in the context of carotenoids production. Haloarchaea represent the group of extremophiles which need high salt concentration to live and develop (even up to 4M of NaCl). Halophilic archaea are able to produce variety types of carotenoids like β-carotene, lycopene or bacterioruberin which may be used both in industry and medicine. Usage of halophilic strains may be a way to reduce costs and to improve quantity and diversity of obtained carotenoids. In this poster we would like to focus on the role of halophilic strains in the production of carotenoids, as well as, their potential role in health improvement and industry. REFERENCES: 1. Calegari-Santos R, Diogo RA, Fontana JD, Bonfim TM. “Carotenoid Production by Halophilic Archaea Under Different Culture Conditions” Curr Microbiol. 2016 May;72(5):641-51. doi: 10.1007/s00284- 015-0974-8. 2. Hou J, Cui HL. “In Vitro Antioxidant, Antihemolytic, and Anticancer Activity of the Carotenoids from Halophilic Archaea” Curr Microbiol. 2018 Mar;75(3):266-271. doi: 10.1007/s00284-017-1374-z. 3. Rodrigo-Baños M, Garbayo I, Vílchez C, Bonete MJ, Martínez-Espinosa RM. “Carotenoids from Haloarchaea and Their Potential in Biotechnology” Mar Drugs. 2015 Aug 25;13(9):5508-32. doi: 10.3390/md13095508.

106 P 53. Diversity of halophilic archaea in food and its link to human microbiome K. Krawczyk, Z. Juszczyk, E. Okła, M. Kowalewicz-Kulbat

Department of Immunology and Infectious Biology, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland Archaea are prokaryotic organisms distinct in the structural and molecular sense from bacteria which inhabit extreme environments. Halophilic archaea have mostly been studied in the environmental fields, but recently, they close relationship with daily human life and microbiome has been discovered. Salting is one of the oldest form of food preservation used by humans. According to the World Health Organization (WHO), sodium consumption for adults should not exceed 2g per day (equivalent of 5g of salt per day), however, the actual consumption is much higher. Fermented, highly salted food is very popular in countries such as South Korea, Thailand and Japan. The presence of halophilic archaea like: Halobacterium and Natrinema (salted- fermented fishery products), Haloarcula marismortui (salted anchovies) or Natronococcus, Natrialba, Halosimplex, Halobiforma and Halococcus genera (kimchi, a Korean fermented food) has been described in many studies on commercial salt or salty products. This work describes the diversity of halophilic strains in salty products and their link to human microbiome. REFERENCES: 1. Lee HS. “Diversity of halophilic archaea in fermented foods and human intestines and their application” J Microbiol Biotechnol. 2013 Dec;23(12):1645-53. 2. Henriet O, Fourmentin J, Delincé B, Mahillon J. “Exploring the diversity of extremely halophilic archaea in food-grade salts” Int J Food Microbiol. 2014 Nov 17;191:36-44. doi: 10.1016/j.ijfoodmicro.2014.08.019. Epub 2014 Aug 20. 3. Gibtan A, Park K, Woo M, Shin JK, Lee DW, Sohn JH, Song M, Roh SW, Lee SJ, Lee HS. “Diversity of Extremely Halophilic Archaeal and Bacterial Communities from Commercial Salts Front” Microbiol. 2017 May 10;8:799. doi: 10.3389/fmicb.2017.00799. eCollection 2017.

107 P 54. Halotherapy as an alternative treatment in medicine K. Krawczyk, Z. Juszczyk, E. Okła, M. Kowalewicz-Kulbat

Department of Immunology and Infectious Biology, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland, Halotherapy is a form of an alternative treatment where salt is used in various forms. Dry salt inhalation reproduces the microclimate of salt caves, with beneficial effect on health. In Poland there are many sites where patients can take advantage of beneficial salty environment, including: natural cave salt excavations, above-ground salt caves, mines or graduation towers like: Wieliczka Salt Mine, Bochnia Salt Mine or Ciechocinek health resort. There are indications (asthma, allergies or cystic fibrosis) and contraindications (tuberculosis, tumours or hyperthyroidism) to take up treatment, which, if not followed, may lead to exacerbation of disease symptoms. The most important guidelines leading to a positive effect is to follow the recommendations concerning the length, the dose and the number of treatments. Improvement of health has long-term effects. In this poster we would like to explain the role of halotherapy as an alternative treatment and to indicate the most important beneficial effects. ACKNOWLEDGEMENTS NCN GRANT 2017/27/N/NZ6/02850 REFERENCES: 1. Zając J, Bojar I, HelbinJ, Kolarzyk E, Owoc A. “Salt caves as simulation of natural environment and significance of halotherapy” Ann Agric Environ Med. 2014; 21(1):.124-127. 2. Pośpiech S, Barucha P, Damijan Z, Błaszczyk J, Czapkowicz-Pośpiech R. “Badania wpływu mikroklimatu podziemnego uzdrowiska w kopalni soli w Wieliczce na masę ciała, zawartość tkanki tłuszczowej i gospodarkę lipidową – doniesienia wstępne” Acta Bio-Optica et Informatica Medica Inżynieria Biomedyczna, vol. 20, nr 4, 2014. 3. Lazarescu H, Simionca I, Hoteteu M, Munteanu A, Rizea I, Iliuta A, Dumitrascu D, Dumitrescu E. “Surveys on therapeutic effects of “halotherapy chamber with artificial saltmine environment” on patients with certain chronic allergenic respiratory pathologies and infectious-inflammatory pathologies” J Med Life. 2014; 7(Spec Iss 2): 83-87.

108 P 55. Salt of flufenamic acid with ethacridine M. S. Krawczyk, I. Majerz

Department of Analytical Chemistry, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wrocław Medical University, Wrocław, Poland An increasing interest in co-crystallization and salt formation of the compound of pharma- ceutical importance results from the possibility of improving of weak solubility and other physical properties as stability or bioavailability. Another reason of co-crystallization of pharmaceutical substances is production of new drugs which exhibit potential pharmaceutical activity including all properties of co-crystal components. Flufenamic acid belongs to the group of fenamic acids that are known as non-steroidal anti-inflammatory substances used in therapy. Obtaining of co-crystals and salts of these group of drugs seems to be an attractive idea of designing and creation of more efficient cures. The synthesis of salt of flufenamic acid with ethacridine that is known as antiseptic agent, may be an approach to compose species in which a synergy between components is expected. The main intermolecular interaction which keeps the molecules of flufenamic acid and ethacridine together in the co-crystal network is intermolecular hydrogen bond. The formation of the network of intermolecular of N–H∙∙∙O, O–H∙∙∙O, N–H∙∙∙F and C–H∙∙∙O hydrogen bonds is observed between components of the salt and water molecules. The crystal structure of flufenamic acid with ethacridine will be presented and analysis of the hydrogen bonds will be performed. ACKNOWLEDGEMENTS The authors thank to Wrocław Medical University for financial support: grant STM.D050.17.002

Fig. 1. The crystal structure of the salt of flufenamic acid with ethacridine.

109 P 56. The influence of insulin gene polymorphisms on its concentration in a selected group of people M. Król1, M. Kepinska2, Ł. Lewandowski2, H. Milnerowicz2

1Students Scientific Association, Department of Biomedical Environmental Analyses, Medical University of Wrocław, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wrocław, Poland 2Department of Biomedical Environmental Analyses, Medical University of Wrocław, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wrocław, Poland [email protected] Insulin is a hormone produced by beta-cells of the pancreas, which is responsible for control of glucose levels in the blood. This protein is encoded by the INS gene, which is located on the shorter arm of chromosome 11.Variations in the insulin gene variable number of tandem repeats (INS VNTR) influenced pancreatic insulin gene transcription and altered insulin secretion. According to the number of repeats, the alleles of INS VNTR are divided into: class I (with 26-63 repeats), class II (with 64-140 repeats), and class III (with 141-209 repeats) [1]. No relationship between the occurrence of INS VNTR class III allele and too low body weight was observed, in a research carried out on prepubertal children born small for gestational age. However, insulin concentration at the 120th minute oral glucose tolerance test was significantly higher in homozygous children with class III, compared to homozygous children with class I [2]. In previous studies, class III/III infants secreted more insulin than I/I infants and heterozygous individuals exhibiting intermediate values [3]. Other studies investigated the association of polymorphisms and glucose, insulin and C-peptide levels during intravenous glucose tolerance test in siblings suffering from type 1 diabetes. No significant differences in blood glucose levels were observed. However, lower insulin and C-peptide levels were observed in I/I homozygotes in post-challenge timepoints [4]. The results showed the relationship between insulin polymorphisms and changes in its secretion and concentration. Specific INS polymorphisms may play a role in the development of insulin resistance. REFERENCES: 1. B. J. Barratt, et al. Diabetes 53(7), 1884-1889, 2004. 2. R. Stawerska, et al. Endokrynologia Polska 67(6), 585-591, 2016. 3. R. A. Bazaes, et al. Clin Endocrinol (Oxf). 59(5), 599-603, 2003. 4. W. Fendler, et al. Pediatr Endocrinol Diabetes Metab. 17(1), 5-9, 2011.

110 P 57. Mixture of MMP-2, MLCK and iNOS inhibitors protects from I/R heart injury in isolated rat heart model A. Krzywonos-Zawadzka1, A. Franczak1, G. Sawicki1,2, A. Olejnik1, I. Bil-Lula1

1Department of Medical Laboratory Diagnostics, Division of Clinical Chemistry, Wroclaw Medical University, Wroclaw, Poland 2Department of Anatomy, Physiology and Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, Canada Introduction: Most of the therapeutic strategies for protection of heart from ischemia/reperfusion injury (IRI) are based on single drug therapies with high concentrations of pharmacological agents. We suggest, that multifactorial basis of injury induced by oxidative stress demands the use of multidrug strategies for the prevention and/or treatment. Thus, the aim of the project was to investigate the effect of use of mixture of low concentrations of iNOS inhibitor with the combination of MLCK and MMP-2 inhibitors to observe a synergistic effect on heart IRI. Material and methods: Hearts extracted from anesthetized male Wistar rats (300-350 g) were perfused using the Langendorff method. After 25 min of aerobic stabilization, hearts were subjected no-flow ischemia (20 min) in the presence or absence of inhibitors mixture (Doxy (1uM), ML-7 (0,5uM), 1400W (1uM)) followed by 30 min of aerobic reperfusion. Next to hemodynamic parameters, biochemical markers of IRI were measured. Results: An increased recovery of contractile function, following I/R, in hearts treated with the a drug mixture was accompanied by decreased degradation of MLC1 (~30%, p=0,057 vs I/R). Decreased affinity of these proteins to MMP-2 degradation resulted from decrease of NO level and hence decreased nitration/nitrosylation of structural proteins. Mixture of inhibitors decreased MMP-2 activity (~30%, p=0,018 vs I/R) and ADMA (~57%, p=0,035 vs I/R) to the level of aerobic control. Conclusions: Combined administration of iNOS, MMP-2 and MLCK inhibitors provides a novel strategy in the prevention of the heart from I/R injury due to modulation of three different pathways of hear injury. ACKNOWLEDGEMENTS This project was funded by the Polish National Science Centre, grant no. NCN2014/15/B/NZ3/04865.

111 P 58. Administration of exogenous iNOS inhibitor protects from ischemia/reperfusion heart injury in rat model A. Krzywonos-Zawadzka1, A. Franczak1, G. Sawicki1,2, A. Olejnik1, I. Bil-Lula1

1Department of Medical Laboratory Diagnostics, Division of Clinical Chemistry, Wroclaw Medical University, Wroclaw, Poland 2Department of Anatomy, Physiology and Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, Canada One of the main component of ischemia/reperfusion injury (IRI) is overproduction of nitric oxide (NO). NO at physiological levels plays an important role in vascular homeostasis while its overproduction is involved in the inflammatory process and metabolism of reactive oxygen species. NO produced during ischemia and oxidative stress may cause specific damage to the myocardium. In order to protect the heart subjected to IRI against the cytotoxic effect of NO, we propose the use of a selective inhibitor iNOS-1400W (N-([3-(aminomethyl)phenyl]methyl) ethanimidamide dihydrochloride). The aim of the project was evaluate the potential cardioprotective effect of 1400W on isolated heart. The isolated rat hearts (male Wistar rats, 300-350g) were perfused with Krebs-Henseleit buffer using the Langendorff method. After 25 min of aerobic stabilization, hearts were subjected to global, no-flow ischemia (20 min) followed by 30 min of aerobic reperfusion in the presence or absence of 1400W (5μM). The haemodynamic parameters of the heart (coronary flow, heart rate, left ventricular developed pressure) were determined. Biochemical markers of IRI were measured in a heart tissue. We have shown that administration of 1400W (5µM) led to full recovery of heart mechanical function in comparison to I/R control (~93,2%, p=0,009 vs IR). Improved heart recovery was associated with decreased production of iNOS (~48%, p=0,012 vs IR), ADMA (~56%, p=0,004 vs IR) and NO (~64%, p=0,03 vs AERO). We confirmed, that administration of 5µM 1400W acts cardioprotective, inhibiting adverse changes in the heart that occurs during acute ischemia and subsequent oxygen shock, during reperfusion of coronary vessels. ACKNOWLEDGEMENTS This project was funded by the Polish National Science Centre, grant no. NCN2014/15/B/NZ3/04865.

112 P 59. Lead toxicity and the concentration of PGE2 and TXB2 in chosen structures of rats brain P. Kupnicka1, E. Metryka1, P. Kapczuk1, D. Simińska1, N. Komorniak2 1Department of Biochemistry, Pomeranian Medical University in Szczecin, Powstańców Wlkp. 72 St, 70-111 Szczecin; 2Department of Biochemistry and Human Nutrition, Pomeranian Medical University in Szczecin, Broniewskiego 24 St, 71-460 Szczecin Introduction: The occurrence of high concentrations of lead compounds in the air of large urban and industrial agglomerations makes it necessary to study their impact on the functioning of living organisms, and among them on the human body. Over the centuries, the approach to lead has changed - formerly lead compounds have been widely used, now they are believed to be a great threat to human health and life, because they affect many important systems in the body, including the immune, excretory, reproductive, digestive, circulatory and nervous system. Toxic effects of lead are manifested above all in the central nervous system. It is known that lead has a significant effect on early and late inflammatory reactions, releasing proin- flammatory, procoagulant and chemotactic factors, subsequently activating macrophages for the production of cytokines and the development of further stages of the immune response. Research on neurotoxic effects of lead has been conducted for at least a decade, however, few studies have been devoted to the pro-inflammatory action of the brain, despite the fact that such effects have been observed in many other cells, tissues and organs. Aim: The aim of the study was to examine the effect of exposure to lead in the pre- and neonatal period on the development of inflammation in the central nervous system. Materials and methods: The studies were carried out on Wistar rats, which received water with lead acetate for two months. The control group consisted of animals receiving tap water. Determination of TXB2 and PGE2 activity in the prefrontal cortex, hippocampus and cerebellum was performed using the enzyme-linked immunosorbent assay (ELISA). The statistical analysis was carried out using Statistica 13. Results: We observed differences between the control group and the study group in TXB2 and PGE2 concentrations. In the study group the concentration of PGE2 was increased and the TXB2 decreased in the prefrontal cortex. In cerebellum the concentration of both analyzed substances was increased in study group. There were no statistically significant differences in the structure of the hippocampus. Conclusuions: The administration of lead influences the development of inflammation in selected brain structures of the young rat. In the past few years, evidence that indicates the important role of inflammatory processes in the pathogenesis of many forms of neurodegeneration has been provided. The results obtained by us suggest that exposure to lead may disturb the functioning of the nervous system, impair cognitive development and may lead to the development of neurodegenerative diseases. REFERENCES: 1. I. Krzywy, E. Krzywy, M. Pastuszak-Gabinowska, A. Brodkiewicz: “Ołów – czy jest się czego obawiac?” 2010, 56, 2, 118-128. 2. Tong S, McMichael AJ, Baghurst PA. “Interactions between environmental lead exposure and sociodemographic factors on cognitive development” Arch. Environ. Health 2000;55:330-335. 3. Canfield RL, Henderson CR Jr, Cory-Slechta DA, Cox C, Jusko TA, Lanphear BP. “Intellectual impairment in children with blood lead concentrations below 10 microg per deciliter” N. Engl. J. Med. 2003;348:1517-1526. 4. A. Skoczyńska, R. Poręba, A. Sieradzki, R. Andrzejak,U. Sieradzka, “Wpływ ołowiu i kadmu na funkcje układu immunologicznego” Medycyna Pracy, 2002; 53; 3; 259-264.

113 P 60. Plant substances in dermatological medications and cosmetics – source of safe bioactive compounds or potential danger? P. Kuś1, F. Kominek1 , A. Chwiećko1, A. Starzec1, M. Brożyna1

1Department of Pharmacognosy and Herbal Medicines, Wroclaw Medical Univresity, Wroclaw, Poland Natural compounds are increasingly used in topical medications as well as in cosmetics that are frequently recommended as a supplementary treatment in various skin diseases. There is a common belief that substances which occur in nature are safer and better tolerated by people than synthetic ones. The INCI (Internatonal Nomenclature of Cosmetic Ingredients) list contains over a thousand natural substances obtained mostly from plants. According to the regulations in the EU (European Cosmetic Directive 76/768/EC) the manufacturers are responsible for the safety of cosmetics, however more than three hundred substances has already been classified as hazardous in the C&L inventory database. The safety assessment of botanicals in cosmetics is difficult mainly because of their variability in composition which depends, among others, on the conditions of growth of the plant, its geographical origin, chemotype or production process itself. The most common adverse reactions against natural substances include immediate and delayed allergies, contact dermatitis and photo-dermatitis. The aim of this work was to analyse factors influencing potential toxicity of botanical ingredients used topically and review those substances which can be hazardous along with adverse effects they provoke.

114 P 61. Voltammetric Determination of Mefenamic Acid on Poly-N-Acetylaniline Modified Glassy Carbon Electrode A. Kwiecień, A. Sroka, I. Majerz

Department of Analytical Chemistry, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wrocław Medical University, Wrocław, Poland Mefenamic acid belongs to Non-steroidal anti-inflammatory drugs (NSAIDs). It is non-selective COX-1 and COX-2 reversible, competitive inhibitor. In human the drug exhibits analgesic and antipyretic activity with relatively limited anti-inflammatory effect. Due to the side effects the main use of mefenamic acid is limited to short-term treatment of primary dysmenorrhea and acute pain [1]. The assay for mefenamic acid may be performed by a titration method (British Pharmacopoeia 2002, European and Indian Pharmacopeias, Pharmacopoeia of the People’s Republic of China) or by a liquid chromatography method (United States Pharmacopoeia 26, Indonesian Pharmacopoeia) [2]. Electroanalytical methods such as voltammetry or polarography are powerful and versatile techniques characterised by high sensitivity, accuracy, precision and large linearity range, with relatively low-cost apparatus [3]. These techniques were used for the determination and assay of a wide range of drugs, often without derivatisation [4]. The choice of working electrode has a huge impact on the sensitivity and reproducibility of analysis. Chemically modified electrodes (CME’s) are quite modern approach in basic electrochemical experiments and chemical sensing. The deposition of quite thin film of certain chemical on the electrode surface endow the CME with desirable chemical or electrochemical properties. In the current work poly-N-acetylaniline (PNAANI) glassy carbon (GCE) modified electrode was used for determination of mefenamic acid in aqueous media. The presentation includes the preparation of chemically modified GCE, studies on electrochemical oxidation of mefenamic acid, effect of pH and scan rate on electrochemical response, linear range and detection limit of the method. REFERENCES: 1. M. Valentovic, “Mefenamic Acid” in xPharm: The Comprehensive Pharmacology Reference, ISBN 978-0-08-055232-3, p. 1-5, Elsevier 2008. 2. H. Poerwono, et. al. Profiles of Drug Substances, Excipients and Related Methodology vol.31, p. 281- 336, 2005. 3. S.A. Ozkan, et. al. Journal of Pharmaceutical and Biomedical Analysis vol. 130, p. 126-140, 2016. 4. N. Abo El-Maali, Bioelectrochemistry, vol. 64, p. 99-107, 2004.

115 P 62. The effect of pH on the optical properties of CdSe / CdS quantum dots modified with cysteamine A. Lesiak1,2, P. Wróbel2, M. Bański2, J. Cabaj1, A. Podhorodecki2

1Faculty of Medicinal Chemistry and Microbiology, Wroclaw University of Science and Technology, Poland 2Faculty of Experimental Physics, Wroclaw University of Science and Technology, Poland A surface applianced an important role in affecting the properties of quantum dots (QDs), including dispersibility, reactivity, stability, melting point or electron structure1. In addition, the surface of the QDs is responsible for their combination with other compounds, e.g. with proteins. Due to this, ligands on the QDs surface are so important for the manufacturing and functioning of QDs-based devices as well as in the coupling of QDs with target molecules e.g. for drug delivery applications. A prerequisite for every possible application is proper QDs surface functionalization2. The ligand exchange process from CdSe/CdS QDs surface from oleic acid to cysteamine has been done. The surface modification under various pH conditions, reaction media (phosphate buffer / deionized water) and with a different alkaline pH stabilizer (NaOH/ tetramethylammonium hydroxide pentahydrate) was aimed at identifying the best conditions for exchange of ligands from the QDs surface. The obtained samples were characterized using absorbance and photoluminescence measurements to confirm the dispersion in an inorganic medium, determine the degree of photoluminescence quenching as a result of the reaction and new properties, i.e. hydrophilicity. ACKNOWLEDGEMENTS The authors gratefully acknowledge the financial support from Wroclaw University of Science and Technology (statutory activity no. 0401/0145/18) REFERENCES: 1. Alivisatos A. P., J. Phys. Chem. C., 100(31), 13226-39, 1996. 2. Sperling R. A., Parak W. J., Philos. Trans. A Math. Phys. Eng. Sci., 368(1915), 1333-83, 2010.

116 P 63. Analysis of the relationship between interleukin-6 promoter polymorphisms and the risk of type 2 diabetes D. Lewoń1, M. Śliwińska-Mossoń2, H. Milnerowicz2

1SKN at Department of Biomedical Environmental Analyzes, Medical University of Wroclaw, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw, Poland 2Department of Biomedical Environmental Analyzes, Medical University of Wroclaw, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw, Poland The etiology of type 2 diabetes (T2DM) has not been fully elucidated, it includes both genetic predisposition and environmental factors, including exposure to cigarette smoke xenobiotics. Interleukin-6 (IL-6) is a multifunctional pleiotropic cytokine involved in regulation of immune responses, acute-phase responses, hematopoiesis, and inflammation. It is produced by endothelial cells, fibroblasts, monocytes, and macrophages in response to different stimuli during systemic inflammation in diseases such as type 2 diabetes [1]. During study in population from north India, two polymorphisms of IL-6 promoter gene were considered to have impact on risk of T2DM: -597 A/G and -174 G/C. PCR-RFLP analysis of 213 patients with T2DM showed no significant association with -597 A/G polymorphism. However, highly significant association was observed with -174 G/C polymorphism. In haplotypic analysis, combination of -597G*/- 174C* showed significant association [2]. Other study investigated the association of IL-6 gene polymorphisms with T2DM and circulating levels of IL-6 in Koreans. There were four polymorphisms included in the study: –174G/C, –572C/G, –597G/A and –1363G/T. Two polymorphisms, –597G/A and –1363G/T, were not detected in examined population and SNP -174 G/C presented very low frequency. The study included 1477 individuals with normal glucose tolerance as a control group and 476 patients with T2DM. The study demonstrated that the IL-6 –572G/G genotype is associated with higher serum IL-6 and hs-CRP concentrations and with increased risk of T2DM [3]. Both studies revealed association between IL-6 promoter gene polymorphisms and a higher risk of type 2 diabetes. This correlation may be used to create screening tests for populations and early modify environmental factors (e.g. smoking) to neutralize risk of type 2 diabetes. The issue of the IL-6 gene promoter polymorphisms is also interesting in terms of significant ethnic or geographical differences in their genetic distribution. REFERENCES: 1. M. Akdis, et al. J. Allergy Clin. Immunol. 138, 984-1010, 2016. 2. M Saxena, et al. Indian J Med Res, 140, 60-68, 2014. 3. S. J. Koh, et al. Clinical Endocrinology, 70, 238–244, 2009.

117 P 64. Sialylation of human milk IgG over lactation J. Lis-Kuberka1 , M. Berghausen-Mazur2 , M. Orczyk-Pawiłowicz1

1Department of Chemistry and Immunochemistry, Wroclaw Medical University, Wroclaw, Poland 2Department of Pediatrics, Division of Neonatology, Wroclaw Medical University, Wroclaw, Poland The human milk is the best nutrient, which ensure the proper growth, development and well- being of newborns. Among a wide range of milk components, glycoproteins, including immunoglobulins, support immature immunological system of newborns and are a part of immunity provided during breastfeeding. In contrast to the most abundant milk S-IgA, the glycosylation pattern of IgG is poorly characterized due to the low concentration. The aim of our work was to study the sialylation profile of milk IgG in relation to milk maturation. The relative amounts of α2,3-/α2,6-sialylated and galacto-/agalactosylated glycotopes on term milk IgG were analyzed by lectin-IgG-ELISA using: Maackia amurensis (MAA), Sambucus nigra (SNA), Ricinus communis I (RCAI) and Griffonia simplicifolia II (GSLII) specific to α2,3-/α2,6-linked sialic acid and Gal(β1,4)GlcNAc-/GlcNAc-, respectively. Milk IgG elicited qualitative and quantitative differences in the sialylation pattern in comparison to plasma IgG of lactating mothers. In contrast to plasma IgG, the expression of α2,3-sialylated glycotopes on milk IgG was observed and it was the highest for mature milk group. The expression of MAA-, SNA- and GSL-reactive glycotopes on milk IgG showed a positive correlation r=0.5, r=0.5 and r=0.3, respectively, with milk maturation, but no correlation was found for the expression of RCA-reactive glycotopes. The presence of highly sialylated glycans of milk IgG might be related to local synthesis in the mammary gland and additional glycans attached to the Fab fragment. Milk IgG sialyl- glycotopes might play an important role in anti-inflammatory activity of milk IgG and moreover, might provide milk IgG with additional binding sites for sialic acid-dependent bacteria.

This work was supported by the Medical Faculty (No STM.A070.17.060), Wrocław Medical University (Poland)

118 P 65. Immune phenomena in the course of treatment with anti-TNFα agents Z.M. Łukasik, J.S. Makowska

Department of Rheumatology, Medical University of Łódź, Łódź, Poland Advances in the understanding of immune pathways involvement in diseases’ pathogenesis brought new therapeutic options and the discovery that seemingly unrelated disorders could share common background. Axial spondyloarthritis (axSpA) is classically perceived as a rheumatic disease of sacroiliac and vertebral joints. In its extreme form axSpA leads to the bony fusion of the spine(1). Inflammatory Bowel Disease (IBD) is a gastroenterological entity, which symptoms often coincide with axial spondyloarthritis. Histological evaluation of intestinal epithelium revealed microscopic gut inflammation in up to 60% of axSpA patients. The severity of gastrointestinal symptoms correlates with radiological changes. Similarly, joint inflammation is a common finding among IBD patients(2). The results of recent studies on the role of mucosal barriers suggested that subclinical bowel inflammation play a role in triggering and perpetuating systemic and joint inflammation in axSpA patients. Tumor necrosis factor α (TNFα) was found to be a key cytokine in the pathogenesis of both axSpA and IBD and is targeted by biological therapies available for both entities(3). However, in primarily rheumatologic patients therapy with anti-TNFα agents sometimes results in unforeseen flares of enterocolitis. This paradoxical effect is observed among primarily gastroenterological patients as well(4). During my presentation I will attempt to elucidate the immune mechanisms leading to the pathogenesis of the disorders as well as explain the immune phenomena occurring in the course of anti-TNFα therapy. REFERENCES: 1. J. D. Taurog, A. Chhabra, R. A. Colbert, “Ankylosing Spondylitis and Axial Spondyloarthritis” N Engl J Med 374, 2563-2574 (2016). 2. M. C. Karreman, J. J. Luime, J. M. W. Hazes, A. E. A. M. Weel, “The Prevalence and Incidence of Axial and Peripheral Spondyloarthritis in Inflammatory Bowel Disease: A Systematic Review and Meta-analysis” J Crohns Colitis 11, 631-642 (2017). 3. V. Ranganathan, E. Gracey, M. A. Brown, R. D. Inman, N. Haroon, “Pathogenesis of ankylosing spondylitis - recent advances and future directions” Nat Rev Rheumatol 13, 359-367 (2017). 4. I. Cleynen, S. Vermeire, “Paradoxical inflammation induced by anti-TNF agents in patients with IBD” Nat Rev Gastroenterol Hepatol 9, 496-503 (2012).

119 P 66. Synthesis and interaction of new meloxicam derivatives with phospholipid bilayers J. Maniewska, B. Szczęśniak-Sięga

Department of Chemistry of Drugs, Wroclaw Medical University, Wrocław, Poland Meloxicam is a a recognized non-steroidal anti-inflammatory drug (NSAID), member of a class of oxicams, used in a treatment of inflammation and pain. The molecular basis of NSAIDs mechanism of action is the ability of these drugs to inhibit cyclooxygenase (COX). COX enzymes are membrane-bound, endoplasmic reticulum (ER) resident, glycoproteins, which catalyse the conversion of arachidonic acid to prostaglandins, acting as second messengers for immune processes and stimulating malignancies. Moreover, a large body of evidence from epidemiological and preclinical studies has shown that NSAIDs have a chemopreventive effect on different types of cancer, especially in colorectal cancer. Furthermore, Lichtenberger et al. have proposed that one of the alternative mechanism by which NSAIDs may induce differential biological effects is their interaction with cellular membranes and an alteration of membrane biophysical properties [1]. The chemopreventive activity of NSAIDs might be at least partially related to their ability to interact with the lipid phase of biological membranes. In order to achieve the main cellular target of NSAIDs which is COX, these compounds have to pass through the biological membranes first. That is why many efforts are being made for better understanding of NSAIDs interaction with biological membranes. In this study, a novel series of potentially biologically active 1,2-benzothiazine 1,1-dioxides– analogues of meloxicam were synthesized. All of the synthesized compounds were subjected to preliminary evaluation for their ability to interact with phospholipid bilayers. To characterize the interaction of newly synthesized NSAIDs analogues with DPPC lipid bilayers the differential scanning calorimetry (DSC) technique was applied. REFERENCES: 1. L.M. Lichtenberger, et al. Biochim Biophys Acta 1821 p. 994-1002, 2012.

120 P 67. Antioxidant and cardioprotective activities of astragalin, a bioactive component of the flowers of horse-chestnut tree (Aesculus hippocastanum L.) P. Marczuk1, J. Kołodziejczyk-Czepas1

1Department of General Biochemistry, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland The use of plant products in the prevention of many diseases is becoming popular. Their biological activity is often associated with antioxidant properties, responsible for the protection against reactive oxygen species (ROS). The phenomenon of negative oxidative stress is related to extensive generation of ROS and impaired or insufficient effectiveness of natural defense systems of organisms. ROS are involved in the pathogenesis of various disorders such as neurodegenerative and cardiovascular diseases or diabetes. Due to this significant contribution of oxidative stress to the pathogenesis and progression of cardiovascular disorders, antioxidant properties of many secondary plant metabolites, protecting against harmful reactions involving free radicals or non-radical ROS, are one of key mechanisms of their health- promoting and cardioprotective actions. Astragalin (kaempferol 3-O-beta-D-glucopyranoside) is a flavonoid, present in the flowers of horse-chestnut tree (Aesculus hippocastanum L.). This work briefly presents the antioxidant and cardioprotective activities of astragalin, based on the available literature. Search for records dealing with antioxidant and cardioprotective actions of astragalin included works indexed in the PubMed, Medline and Science Direct as well as international and national scientific periodicals, not covered by these databases. Both in vitro and in vivo works demonstrated antioxidant effects of astragalin. Its antioxidant and cardioprotective activity was confirmed by several basic and animal studies, and include its inhibitory activity on the autophagy-associated airway epithelial fibrosis, protective effects against ischemic brain injury as well as amelioration of acute reperfusion-induced injury. The work is supported by a grant from University of Lodz, Poland (506/1136).

121 P 68. Evaluation of cyanine dyes as potential photosensitizers D. Marchewka1, A. Chwiłkowska2

1 Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, Borowska 211 A St., 50-566 Wroclaw, Poland; 2Department of Molecular and Cellular Biology, Medical University, Borowska 211 A St., 50-556 Wroclaw, Poland; Photodynamic reaction (PDR) is utilized in anticancer treatment as it induces cytotoxicity through generation of reactive oxygen species (ROS) due to excitation of photosensitizers. The aim of the work was to experimentally learn the biological activities of cyanines and their photosensitizing properties in vitro. The experiments were carried out on the A549 line (human lung adenocarcinoma line) and primary cell cultures of fibroblasts. The absorption spectra of the substance (1. IR-775 chloride, 2. Zinc phthalocyanine, 3. 5,10,15,20-Tetrakis-21H, 23H- manganese (III) chloride porphine, 4. Aluminum phthalocyanine chloride, 5. Photofrin as a reference) was performed. The cytotoxicity of the compounds, followed by of distribution of the compounds in the cells was evaluated. Tested cyanines showed promising biological activities as potential photosensitizers.

122 P 69. Magnetosomes as a new drug delivery system in cancer therapy A. Maruszewska1, W. Żwierełło2, D. Kuzajewska1

1Department of Biochemistry, Faculty of Biology, University of Szczecin, Felczaka 3c, Szczecin 71-412, Poland 2Department of Biochemistry and Human Nutrition, Pomeranian Medical University in Szczecin, Broniewskiego 24, Szczecin 71-460, Poland An important clinical problem in contemporary medicine is a significant increase in cancer cases and high mortality among patients. One of the reasons is the low effectiveness of conventional anticancer therapies resulted from various mechanisms of tumour cell resistance for treatment. In this context, a significant challenge for the biological and medical sciences is the development of new, more effective forms of therapy. Some microorganisms are associated with the oncogenesis. On the other hand, they can be used as a potential tool in the treatment of cancer. In this aspect, magnetotactic bacteria (MTBs) show significant application potential. MTBs are commonly found in water reservoirs and their sediments. MTBs, due to the presence of magnetosomes, exhibit magnetotaxis. Magnetosomes are organelles containing magnetic minerals (magnetite and greigite) surrounded by a biological membrane. More and more studies indicate that these non-pathogenic bacteria and their biocompatible organelles may be used as carriers of drugs, toxins or gene constructs directed against cancer cells. In addition, magnetosomes, as ferromagnetic structures, may serve not only as a therapeutic agent (through magnetic hyperthermia or photothermia), but can also be used in the diagnosis and monitoring of cancer. MTBs and magnetosomes are therefore a promising tool in cancer therapy, and the research results on MTBs are important reasons for further work on this group of microorganisms.

123 P 70. Inflammatory response of macrophages of the THP 1 cell line to environmental lead concentration E. Metryka1, P. Kupnicka1, P. Kapczuk1, D. Simińska1, D. Styburski2, M. Bosiacki3

1Department of Biochemistry, Pomeranian Medical University in Szczecin, Powstańców Wlkp. 72 St, 70-111 Szczecin; 2 Department of Biochemistry and Human Nutrition, Pomeranian Medical University in Szczecin, Broniewskiego 24 St, 71-460 Szczecin, 3Department of Medical Rehabilitation and Clinical Physiotherapy, Pomeranian Medical University in Szczecin, Żołnierska 54 St, 71-210 Szczecin Introduction: The rapidly growing industry contributes to the emergence of new threats with which the human immune system has to face. Lead (Pb) is a widespread pollutant and the presence of this element in the environment is associated with the problem of harmful effects of even low Pb doses. The constant presence of Pb compounds makes it necessary to study their effect on the functioning of organisms. Macrophages are a very important element of a well- functioning immune system and it seems justifiable to check how exposure to Pb affects their function. Aim: The aim of the study was to test the influence of Pb on the functions of macrophages taking into account the pro-inflammatory properties of the examined element. Materials and methods: The tests were performed on THP-1 cells. It is a widely used research model in experiments to study the physiology or pathology of human macrophages. The cells were grown in RPMI 1640 medium supplemented with 10% FBS, 100IU/ml penicillin and 10μg/ml streptomycin at 37°C, 5% CO2 and 95% humidity. Monocytes were differentiated to macrophages with PMA. The cells were subjected to 24 h incubation with lead acetate, with a final concentration of lead: 1.25μg/dL; 2.5μg/dL; 5μg/dL; 10μg/dL. Control groups were cultured without the addition of an irritant or with the addition of sodium acetate (10μg/dL). The concentration of TXB2, PGE2, IL1 and IL6 in the culture medium was determined by the colorimetric method. Results: Statistically significant differences were observed in each of the analyzed proinflammatory factors. Incubation of cells with Pb at a concentration of 5μg/dL resulted in an increase in the amount of PGE2 relative to the groups treated with 1.25μg/dL; 2.5μg/dL Pb and control and also increase in TXB2 concentration compared to controls. The increase in IL1 was demonstrated in the group exposed to 10μg/dL Pb in relation to 1.25μg/dL Pb and control. The increase in IL6 concentration after incubation with Pb at concentrations of 5μg/dL and 10μg/dL was observed in relation to the group exposed to 2.5μg/dL Pb. Conclusuions: The obtained results suggest that the intensity of the inflammatory response is dependent on the concentration of lead administered to the cell cultures. REFERENCES: 1. Baranowska-Bosiacka, I., Kosińska, I., Jamioł, D., Gutowska, I., Prokopowicz, A., Rębacz-Maron, E., I inni. 2016. “Environmental Lead (Pb) Exposure Versus Fatty Acid Content in Blood and Milk of the Mother and in the Blood of Newborn Children” Biological Trace Element Research. 2016, pp. 279-87. 2. CDC. 2012. Low Level Lead Exposure Harms Children: A Renewed Call for Primary Prevention. Report of the Advisory Committee on Childhood Lead Poisoning Prevention of the Centers for Disease Control and Prevention. 2012. 3. Nazimek K. i Bryniarski K. 2012. “Aktywność biologiczna makrofagów w zdrowiu” Postępy Higieny I Medycyny Doświadczalnej (online). 2012, pp. 507-520.

124 P 71. Pro-health properties of polyphenic extract of Cistus Incanus in prevention of arteriosclerosis K. Męczarska1, S. Cyboran-Mikołajczyk1, D. Bonarska-Kujawa1, A. Włoch1, J. Oszmiański2

1Department of Physics and Biophysics, Wrocław University of Environmental and Life Sciences, Wrocław, Poland 2Department of Fruit, Vegetable and Cereal Technology, Wrocław University of Environmental and Life Sciences, Wrocław, Poland [email protected] One of the most common diseases of 21st century society is atherosclerosis, which results in ischaemia and hypoxia of many organs. Numerous studies have shown that plant-derived compounds are characterized by good bioavailability. One of such substances is Cistus Incanus extract, which due to its high content of polyphenolic substances exhibits strong anti- inflammatory, antibacterial and antiviral activity. Pro-health properties of Cistus Incanus encouraged the authors to conduct biophysical studies on the influence of its polyphenol extract in the prevention of atherosclerosis. Objective of the research was to determine the biological activity of two fractions of polyphenolic compounds extracted from Cistus Incanus in relation to erythrocytes treated as an example and cell model and their membranes adopted as a model of biological membranes. The spectroscopic study confirmed high antioxidant activity of the polyphenols contained in Cistus Incanus extract, compared to that of: Myricitin and Trolox®. These compounds have no toxic effects on erythrocytes because they do not cause their hemolysis. The extracts used induce changes in erythrocyte shapes and significantly increase the resistance of red blood cells to changes in osmotic pressure. These extracts effectively protect erythrocytes against oxidation induced by both AAPH compound and UVC radiation. Polyphenolic substances contained in examined extract, having a high ability to scavenge and neutralize free radicals, can effectively protect cells and their components against oxidative damage, thus delaying the aging process. Therefore, they can prevent the development of many diseases caused by oxidative stress, as well as be used in the prevention of atherosclerosis. ACKNOWLEDGEMENTS Work financed from funds on the statutory activity of the Department of Physics and Biophysics of Wrocław University of Environmental and Life Sciences.

125 P 72. 1H NMR analysis of Pseudomonas aeruginosa K. A. Mielko1, D. Sands2, S. Jabłoński3 M. Łukaszewicz3, P. Młynarz1

1Bioorganic Chemistry Group, Faculty of Chemistry, Wroclaw University of Science and Technology, Wroclaw, Poland 2Mother and Child Institute, Warszawa, Poland 3Biotransformation Department, University of Wroclaw, Wroclaw, Poland Pseudomonas aeruginosa is the most common pathogen, which occurs in the patients who suffer from respiratory tract of cystic fibrosis (CF). In a chronic infection, this bacterium is practically impossible to eradicate because of its high resistance for antibiotics treatment. This fact is associated with increased morbidity and mortality rate. Although, an early infection may be easier to combat, but the performed diagnostics usually is too late [1]. In the last years NMR (nuclear magnetic resonance) has become one of the primary methods used for metabolomics studies. Metabolomics involves an analysis of metabolic profiles of microbial extracts (intra and extracellular) using NMR spectroscopy and MS spectrometry methods coupled with multivariate data analysis. NMR is used in microbial metabolomics because it is nonselective method in metabolite detection, quantitative, reproducible and rich in structural information. In addition to transcriptome and proteome studies, metabolome analysis represents a third complementary approach to identify metabolic pathways and adaptation processes [2, 3]. The main goal of this study was to compare metabolomics profile of two different groups of Pseudomonas aeruginosa – clinical strains isolated from cystic fibrosis patients and strains isolated from the environment. Moreover, differentiating of the metabolites was identified like: alanine, leucine, isoleucine, valine, glutamate, betaine, lactate, acetate, ethanolamine, glycerol and glycine. Principal component analysis PCA of the obtained data revealed that these two groups can be distinguished on the basis of metabolomic profile. REFERENCES: 1. C. Winstanley at al., “Pseudomonas aeruginosa Evolutionary Adaptation and Diversification in Cystic Fibrosis Chronic Lung Infections” Trends in Microbiology, 24(5), 327-337, 2016 2. B. B. Aldridge et al., “Microbial metabolomics: Innovation, application, insight” Current Opinion in Microbiology, 19(1), 90-96, 2014 3. S. Barnes et al., “Training in metabolomics research. II. Processing and statistical analysis of metabolomics data, metabolite identification, pathway analysis, applications of metabolomics and its future” Journal of Mass Spectrometry, 51(8), 535-548, 2016

126 P 73. Isothiocyanates M. Milewicz

Institute of Immunology and Experimental Therapy, Department of Experimental Oncology, Wrocław, Poland In recent years cancerous diseases have become a great, global problem. According to data published by WHO, 9.6 million people died from cancer in 2018 placing it as a second most common cause of death [1]. Many studies show the importance of diet in the prevention and therapy of numerous diseases also including cancer. Special attention should be paid to cabbage plants. It is proved that compounds present in these plants have the ability of interaction with the metabolic pathways involved in each stage of cancer development [2, 3]. Noteworthy are glucosinolates - isothiocyanate precursors (ITC). ITCs influence both the carcinogenesis process and early stages of cancer promotion, but also the stage of rapid tumor growth, angiogenesis or even metastasis [3]. These compounds have a potential to be ideal candidates as drugs, but many adversities are on the way of the development of effective therapies. Namely, ITCs do not possess any specific molecular target. At least 30 proteins are known to be directly modified by them. In addition, ITCs also have an indirect effect on cell functions. This makes it difficult to thoroughly investigate the mechanisms of ITCs biological activity, which is considered as their main disadvantage [4]. However, taking into account that each of the neoplastic diseases has its own specificity, and the development involves numerous complex biochemical pathways, the lack of a precisely defined target should be considered as an advantage of ITCs. REFERENCES: 1. „Cancer”. [Online]: https://www.who.int/news-room/fact-sheets/detail/cancer. [Seen: 08-sty-2019]. 2. G. Murillo and R. G. Mehta, at Nutr. Cancer, t. 41, nr 1-2, s. 17-28, 2001. 3. L. Mi, A. J. Di Pasqua, i F.-L. Chung, Carcinogenesis, t. 32, nr 10, s. 1405-1413, 2011. 4. S. V. Singh i K. Singh, Carcinogenesis, t. 33, nr 10, s. 1833-1842, 2012.

127 P 74. Effects of Pelargonium sidoides root extracts on the growth and activity of alkaline phosphatase in cultures of human mesenchymal stem cells O. Miłek1, T. Męcik-Kronenberg2, G. Tylko1, A. M. Osyczka1

1Department of Cell Biology and Imaging, Institute of Zoology and Biomedical Research, Jagiellonian University, Kraków, Poland 2Department of Pathomorphology, Medical University of Silesia, Katowice, Poland Pelargonium sidoides is a medicinal plant growing in popularity and known for its antibacterial and antiviral properties. Recently, the potential of P. sidoides extracts to treat periodontitis and regenerate surrounding tissues has been postulated. This study examined the effects of P. sidoides root extracts (PSRE) and proanthocyanidins from these extracts (PCAN) on the cell growth and activity of alkaline phosphatase (ALP) in human mesenchymal stem cell (MSC) cultures. MSC are multipotent cells capable of regenerating bone at the site of its damage. During osteoblast differentiation, these cells increase ALP activity, which results in the formation of inorganic phosphorus, important for the bone mineralization process. The tested substances were added to cultures of stem cells derived from bone marrow (BMSC), dental pulp (DPSC) and adipose-derived mesenchymal stem cell line (ASC52telo). PCAN and PSRE were dissolved in medium containing MEM Alpha, 10% MSC-qualified fetal bovine serum and 1% antibiotics to obtain concentrations of 0.0005%, 0.001%, 0.005% and 0.01%. Cells were treated with the tested substances in a standard culture medium and in the medium supplemented with osteoinductive factors: ascorbate-2-phosphate and dexamethasone. The experiments were carried out for 7 days. Cell viability was then measured using MTS assay and ALP activity was examined with p-nitrophenyl phosphate as a substrate. PCAN reduced the viability of all cell types; however, ALP production was increased in bone marrow and dental pulp stem cells. PSRE also reduced the viability of bone marrow and dental pulp stem cells, but to a lesser extent than PCAN. Increased ALP activity in response to PSRE was also observed in dental pulp stem cells cultured in standard medium without osteoinductive factors. Due to the significant influence of P. sidoides root extracts on DPSC, it can be concluded that the surviving subpopulation of DPSC is resistant to the cytotoxicity of PCAN and PSRE and differentiates towards osteoblasts in response to the tested substances. ACKNOWLEDGEMENTS This study is financed in part by grants UMO-2016/22/Z/ST5/00692 (GT, AMO) and UMO- 2016/21/B/NZ5/00217 (AMO)

128 P 75. Cytotoxic potential of extract of Moringa Oleifera in human breast cancer cells P. Młynarczykowska1, N. Rembiałkowska2

1Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, Borowska 211 A St., 50-566 Wroclaw, Poland 2Department of Molecular and Cellular Biology, Medical University, Borowska 211 A St., 50-556 Wroclaw, Poland The available research indicate that Moringa Oleifera has many biological activities and therapeutic properties. It can be mentioned the following features: antihypertensive, diuretic, cholesterol lowering, antispasmodic, anti-inflammatory, antimicrobial and much more activities. Chemical analysis showed that the extract from this plant contains important minerals, amino acids, proteins, vitamins, β-carotene, phenolics. All these constituents have a huge influence on human health. In the past moringa has been used in folk medicine and now it becomes also a popular diet supplement. Regarding to above reasons in this study we focused mainly on antiproliferative and cytotoxic properties. The aim of analysis was to measure the cytotoxic effect of isopropyl isothiocyanate – an active substance, which is known as an anticancer compounds of Moringa Oleifera. We used MCF-7/WT breast cancer cells as a model in vitro. The cytotoxicity was evaluated after 24 and 72 hours by the MTT and SRB assay. The obtained results suggest that the moringa extract exhibits cytotoxic properties against human breast cancer in vitro. In the future it might be used in anticancer treatment. ACKNOWLEDGEMENTS The research was financed partially by the Student Scientific Group “Biology of cancer cells” (Wroclaw Medical University) and National Science Center, Poland, grant No. DEC- 2015/19/N/NZ7/01105 (PI: N.Rembiałkowska).

129 P 76. Tripterine induces DNA double-strand breaks in colon cancer cells H. Moreira1, A. Szyjka1, E. Barg1

1Department of Basic Medical Sciences, Wroclaw Medical University, Wroclaw Tripterine (Celastrol) is one of the most biologically active compound isolated from Tripterygium wilfordii. It has been found to exhibit strong anticancer activities in various cancer models, e.g. inhibition of tumor proliferation and growth as well as tumor capacity to metastasis. However, the action of tripterine on DNA double strand breaks (DSBs) in colon cancer cells remains unknown. The DNA DSBs are strongly deleterious and harmful DNA damage which can lead to cell cycle arrest or induction of apoptosis. In this work, we investigated the effect of Tripterine on the level of phosphorylated H2AX histone (γH2A.X), a biomarker of DSBs, in association with cellular response to DNA damage (cell cycle and apoptosis) in colon cancer cells. Human colon cancer cells: sensitive (LOVO cell line) and resistant (LOVO/DX cell line) to cytotoxic drugs were incubated for 4 or 18hrs with tested plant [1, 2.5, 5μM]. The percentage of γH2A.X positive cells was evaluated using antiphospho-Histone H2A.X (Ser139)-FITC mAb (Invitrogen). The cell cycle analysis was done by PI or DAPI staining method. The rate of apoptotic cells was estimated with Dead Cell Apoptosis Kit (Thermo Fisher). Flow cytometric measurements were carried out with CyFlow®Space cytometer (Sysmex-Partec), and data were analysed using FlowMax software and MultiCycleTM DNA analysis model (FCS 4 Express, DeNovo Software). Our results show that Celastrol significantly increases γH2A.X+ frequency in both LOVO and LOVO/DX cells. The percentage of γH2A.X+ positive cells in LOVO cell cultures was 5-29% and in LOVO/DX cells was 1.9-21%, depending on concentration. The level of spontaneous DBS breaks in control cells was 1.4% (LOVO) and 1.2% (LOVO/DX). These results correlate with celastrol - induced increase in the percentage of cell undergoing apoptosis, mainly in late apoptosis, as well as with cell cycle arrest, i.e. accumulation of cells occupying the S phase of the cell cycle together with marked decrease of cell population in the G2/M phase. γH2A.X+ positive cells were observed in all phases of the cell cycle, however S phase cells revealed a lower frequency of γH2A.X+ cells compared to G1 and G2/M phase cells. These observations did not differ between LOVO and LOVO/DX cells. Celastrol induces DNA DSBs in colon cancer cells which results in the expression of γH2A.X+ throughout all phases of the cell cycle. The celastrol – induced DNA damage leads to inhibition of cell proliferation by cell cycle arrest and induction of apoptotic cell death, both in drug sensitive and drug-resistant colon cancer cells. These results strongly suggest the potential use of this compound in the treatment of colon cancer at various stages of the disease.

130 P 77. Human serum may influence the expression of Yersinia sp. virulence-associated genes K. Morka1, J. Schubert2, E. Wałecka-Zacharska2, E. Klausa3, J. Bystroń2, J. Bania2, G. Bugla-Płoskońska1 1Department of Microbiology, Institute of Genetics and Microbiology, University of Wrocław, Wrocław, Poland 2Department of Food Hygiene and Consumer Health Protection, Wrocław University of Environmental and Life Sciences, Wrocław, Poland 3Regional Centre of Transfusion Medicine and Blood Bank, Wrocław, Poland Y. enterocolitica belonging to Yersinia genus is responsible for both intestinal and parenteral infections. An asymptomatic bacteremia or sepsis occur due to bacterial serum resistance. Therefore, the aim of this study was to evaluate the changes in expression of yadA, ail, and ompC in response to normal human serum (NHS) treatment. Relative expression of selected genes using RT-qPCR was assessed on serum: resistant (3d, 58d, 90z, 169z, 4dz), moderate resistant (10dz) and sensitive (121z, 205dz) strains, in two conditions, with NHS and heat inactivated NHS (iNHS) [1]. The level of serum sensitivity was assessed based on the CFU/ml after 1 hour incubation in 50% NHS relative to the initial number of bacteria. In serum-resistant group increased expression of virulence factors after NHS-treatment at least at one time point during the experiment was shown in 2, 3 and 4 out of 5 strains for ail, yadA and ompC, respectively. Serum-sensitive strains expressed just 1 gene at higher level, i.e. ompC and ail in 121z and 205z, respectively. However, their expression even increased in bacteria cultured in iNHS. At least one virulence-associated gene was demonstrated to have a higher expression after NHS-treatment in majority of tested strains, except 10dz strain, which did not express ompC at higher level. Yersinia sp. serum resistance is not conferred by a single gene and other factors might be important in this phenomenon, what has been observed for 10dz or 205dz strains. ACKNOWLEDGEMENTS This work was supported by special-purpose grant of the Ministry of Science and Higher Education to carry out research or development work and tasks related to the development of young scientists and doctoral participants [Faculty of Biological Sciences, University of Wroclaw, 0420/2562/18]. REFERENCES: 1. J. Schubert, et al. Int. J. Food Microbiol., 235, p. 36-45, 2016.

131 P 78. Optimization of solid lipid nanoparticles (SLN) with natural soy phosphatidylcholine as a tool for oral treatment N. Niezgoda1, E. B. Souto2, J. Kulbacka3, A. Gliszczyńska1

1Department of Chemistry, Wrocław University of Environmental and Life Sciences, Wrocław, Poland 2Department of Pharmaceutical Technology, University of Coimbra (FFUC), Coimbra, Portugal 3Department of Molecular and Cellular Biology, Medical University of Wrocław, Wrocław, Poland Lipid nanocarriers are promising tool for delivery of lipophilic drug enhancing their bioavailability and they are employed in pharmaceutics and biomedical formulations [1]. This study was focused on the optimization of the composition of solid lipid nanoparticles (SLN) with natural soybean phosphatidylcholine (SBPC) as a component of surfactant phase to obtain stable nanoparticles dispersion using the high shear homogenization (HSH) method followed by ultrasonication. To define optimal formulation of SLN a 23 full factorial design were used. Three independent variable were as follows: percent of lipid phase (Gelucire® 43/01), percent of soy phosphatidylcholine (Lipoid® S 100), percent of polysorbate 80 (Tween® 80). The effects of the SLN composition on the mean particle size, polydispersity index and zeta potential were tested. SLN were produced using 24,000 rpm HSH for 2 minutes in 55oC followed by ultrasonication for 3 minutes. Morphology was determined by TEM and revealed fairly spherical shape of NP. SLN were subjected to stability study and were stored either in 4, 20 or 40oC for 2 and 7 days. Lumisizer® analysis showed creaming phenomenon in SLN formulation. Sample stored in 4oC were bigger in size and polydispersity but also more stable during centrifugation. Instability index were below 0,35 for samples stored either for 2 and 7days. This factorial design study allowed to obtain SLN with small diameter (<200 nm), low polydispersity (<250) potentially suitable for effective internalization in cells. The present results proved the usefulness of applying statistical design method in the optimization and preparation of lipid nanoparticles. ACKNOWLEDGEMENTS Studies were supported by Wroclaw Centre of Biotechnology, programme The Leading National Research Centre (KNOW) for years 2014-2018. REFERENCES: 1. P. Ganesan, et al. Sustainable Chemistry and Pharmacy, vol. 6, p. 37-56, 2017.

132 P 79. Klotho protein - from Greek mythology to cardioprotection? A. Olejnik1, A. Krzywonos-Zawadzka1, M. Banaszkiewicz1, I. Bil-Lula1

1Division of Clinical Chemistry, Department of Medical Laboratory Diagnostics, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, Wroclaw, Poland Klotho protein is associated with aging and the name of gene comes from Greek mythology, which says that Clotho, one of the Three Fates, was responsible for spinning the thread of human life. The aim of the study was to evaluate the role of Klotho in the ischemia/reperfusion (I/R) injury of cardiac cells. Isolated rat hearts and human cardiac myocytes (HCM) were subjected to aerobic and I/R conditions. The expression of Klotho protein in the hearts and its secretion into coronary effluents, as well as surface expression of Klotho in HCM were assessed. HCM were incubated in the presence of recombinant Klotho protein and the viability of cells was measured. Klotho protein was expressed in rat hearts and HCM, in both aerobic and I/R conditions. There was a significant increase in Klotho levels in coronary effluents in I/R group (p<0.05). Klotho levels in coronary effluents positively correlated with lactate dehydrogenase activity (marker of cardiac injury) (p<0.05, r = 0.6) and negatively correlated with recovery of rate pressure product (grade of cardiac mechanical function) (p<0.05, r = -0.6). The cell surface expression of Klotho in HCM was significantly higher in I/R group (p<0.05). The treatment of HCM with recombinant Klotho resulted in increased viability of cells after I/R (p<0.05). An increased expression of Klotho protein in I/R HCM and increased viability of these cells suggests a potential compensatory or cardioprotective effect. The release of Klotho into the extracellular space indicates the utility of Klotho as a marker of cardiac damage.

This work was supported by the National Science Centre [grant number UMO- 2016/23/NZ3/03151].

133 P 80. When pharmaceuticals meet the microbiome – the importance of pharmacomicrobiomics M. Ogorzałek1, P. Piątkowska1, E. Sawicka2

1 Student Group of Toxicology, Wroclaw Medical University, Wroclaw, Poland 2 Department of Toxicology, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, Wroclaw, Poland Human microbiome is an intriguing area in modern medicine. In order to better understand the concept of intestinal microbiome, it becomes clear that its composition is influenced by many other drugs besides antibiotics, but also microflora can change the effectiveness of xenobiotics and contribute to individual reactions to pharmacotherapy. These interactions are defined by the term "pharmacomicrobiomics". One of the mechanisms underlying the metabolic role of microflora is production of secondary bile acids by certain intestinal bacteria. These molecules interact with FXR receptors, which activation affects the expression of drug-metabolizing cytochrome P450 isoenzymes. So far, about 40 xenobiotics have been reported whose metabolism has been affected by microbiota, both directly and indirectly. Pharmaceuticals co-metabolized by intestinal bacteria include, e.g., L-dopa, indomethacin, nitrazepam and loperamide. Microbiome may play a role in the individual variability of the bioavailability and efficacy of simvastatin and lovastatin. There is a correlation between the susceptibility to acetaminophen-induced hepatotoxicity and the level of microbial metabolite - p-cresol, because they can compete for cytosolic sulfotransferase. On the other hand, many pharmaceuticals can change the microbiota, including antibiotics, PPIs and NSAIDs, which reduce the diversity of microflora, affecting the physiology and gene expression of active intestinal bacteria. Elucidating microbe-drug cometabolic pathways could yield new therapeutic options for human health, contribute to personalized medicine development and improve the rational drug design. Targeting drugs to microbial genes and modifying the microbiota can be potentially used in treatment and modifying many human disease risk factors, such as T2D and IBD.

134 P 81. Evaluation of antioxidant capacity and polyphenol content of selected vegetable species using the alcoholic and buffer extraction R. Olędzki1,2 , U. Kaim1,2, A. Orkusz1,2, L. Bogacz-Radomska1,2 , J.Harasym1,2

1 Adaptive Food Systems Accelerator – Science Centre, Wrocław University of Economics, Poland 2 Department of Biotechnology and Food Analysis, Institute of Chemistry and Food Technology, Wrocław University of Economics, Poland Vegetables are considered an valuable source of antioxidants and bioactive substances which are efficient in protecting the human body against the reactive oxygen species. The objective of this study was to assess the polyphenol content and antioxidant activities of two species of vegetables – parsley and kohlrabi. The specific parts of vegetables tested (root or bulb and leaves) were subjected to chemical extraction using ethanol, methanol and buffer. Polyphenol content, antioxidant capacity and ferric reducing ability of different parts of vegetables were determined by Folin-Ciocalteu, ABTS and FRAP methods. It was observed that polyphenol content obtained with all used extraction methods was significantly different both between species and parts of vegetables (the leaves of the investigated vegetables contained more polyphenols than the storage tissues of this plants - bulb and root). Ferric reducing ability was also significantly different in the tested extracts obtained by all using methods, with higher values in extracts from the leaves of the investigated vegetables (the highest reducing power occurred in the parsley leaves). The highest value of antioxidant capacity in tested vegetables was in the ethanol extracts obtained from the parsley leaves. Polyphenol content, antioxidant capacity and ferric reducing ability were also significantly different in the extracts obtained by the all used methods, with higher values in alcoholic extracts. All the obtained results are also revealed the important differences related to extraction method as well as the species of vegetables. Additionally, the buffer extraction was the most effective in the case of parsley root.

135 P 82. The content of total polyphenols and antiradical activity in different green leafy vegetables grown in Lower Silesia area in Poland R. Olędzki1,2, U. Kaim1,2, A. Orkusz1,2, L. Bogacz-Radomska1,2 , J. Harasym1,2

1Adaptive Food Systems Accelerator – Science Centre, Wrocław University of Economics, Poland 2Department of Biotechnology and Food Analysis, Institute of Chemistry and Food Technology, Wrocław University of Economics, Poland Green leafy vegetables eating in form of salads belongs to the group of foods that are consumed by humans regularly and in relatively large quantities, especially in the spring and summer season. Pro-health values of such vegetables can be determined by the content of vitamins and minerals, as well as by the antiradical activity and content of bioactive components from the group of polyphenols. Various leafy vegetables are a rich source of polyphenols and other bioactive compounds. The scientific research confirms the special role of polyphenols in the prevention of civilization diseases. The aim of this study was to analyze the 6 most popular green leafy vegetables in Poland, according to bioactive properties: the polyphenols content and antiradical activity. The content of total polyphenols was determined by the Folin-Ciocalteu method. Antiradical activity was determined by DPPH methods. The highest content of total polyphenol were observed for rucola, red and lamb's lettuce. The lowest contents of total polyphenols were measured in romaine and butterhead lettuce. The highest values of antiradical activity were determined in variety red and lamb's lettuce. The lowest values of antioxidant activity was determined in variety butterhead and iceberg lettuce. A significant varietal dependence was demonstrated in lettuce leaves taken from individual varieties of lettuce. The total polyphenols content and antiradical activity of the lettuces can be influenced also the location of plant cultivation in Lower Silesia. Based on the conducted tests, it can be concluded, that lettuces should find its place in our diet.

136 P 83. Polymorphism of metallothionein 2A (rs10636) in relation to pathological processes M. Ołdakowska, H. Milnerowicz

Department of Biomedical and Environmental Analyses, Faculty of Pharmacy, Wroclaw Medical University, Poland Metallothioneins (MTs) are low molecular weight cysteine-rich proteins family with a capability to bind transition metal ions with the highest affinity to copper, cadmium and zinc [1]. MTs are encoded by genes located on chromosome 16q13 and have four main isoforms MT1, MT2, MT3 and MT4 [2]. MTs take part in metals homeostasis, protect against heavy metal toxicity, DNA damage and participate in cellular defense mechanisms against oxygen free radicals [3]. The aim of the study is an overview of the contemporary reports about influence of polymorphism of metallothionein 2A gene on pathological process in organism. The human genome contains iterative forms of structural genetic variations. Single nucleotide polymorphisms (SNPs) are the most common type of them in DNA sequence and occur in greater than one percent of the population [4]. SNPs generate changes in DNA sequence, in result may cause change amino acids, conformation and structure of proteins and may affect on molecules and ions balance [5]. Recent investigations show that MT2A polymorphisms associated with metal ions homeostasis. Polymorphism rs10636 significant decrease intracellular and serum in both zinc and copper levels [6]. Genetic variation of MT2A is correlated with morbidity of the diseases related to inappropriate lifestyle such type 2 diabetes mellitus, diabetes neuropathy, hyperlipidemia and atherosclerosis [7]. Beside, polymorphism rs10636 may contribute to induce many type of cancer, activate inflammatory status and NK cell cytotoxicity [8]. Polymorphism in metallothionein 2a gene could be related with many pathological processes. REFERENCES: 1. J. Calvo, et al. IUBMB Life, vol.69, issue 4, 2017. 2. M. Si et al. Journal of Hematology & Oncology, 11, 1, 2018. 3. G. Isani et al. Biomolecules, 4, 435-457, 2014. 4. J. Wu, FEBS Letters, 586, 2841-2845, 2012. 5. C. Schaefer et al. BMC Genomics 13(Suppl 4):S4, 2012. 6. M. Raudenska et al. Metallomics, 6, 55-68, 2014. 7. L. Yang et al. Am. J. Physiol.: Endocrinol. Metab., 294, E987–E992, 2008. 8. R. Giacconi et al. Mol. Med., 13, 388-395, 2007.

137 P 84. Evaluation of the diet of children aged 4-6 in a chosen kindergarten in Wrocław A. Orkusz1,2, U. Kaim1,2, R. Olędzki1,2, L. Bogacz-Radomska1,2, J. Harasym1,2, M. Dyrcz2

1Adaptive Food Systems Accelerator – Science Centre, Wrocław University of Economics, Poland 2Department of Biotechnology and Food Analysis, Institute of Chemistry and Food Technology, Wrocław University of Economics, Poland Nutrition is one of the most important factors assuring normal growth and development of children. Proper children nutrition reduces the risk of excessive body weight, and consequently prevents many diseases caused by inadequate nutrition. It should be remembered that children during the period of growth show a special sensitivity to any nutritional deficiencies that arise during this period. More and more research projects are indicating that there is a close relationship between the state of nutrition during childhood and the health of an adult. The aim of this study was the energetic and nutritional value evaluation of meals served in the chosen kindergarten in Wrocław based on theoretical analysis of the menus using the Dietician 2 computer program. Energetic value, content of basic nutrients (protein, fat and carbohydrates), fiber, anti-oxidative vitamins (A and C) and selected mineral ingredients (Ca and Fe) were calculated for kindergarten menus for four seasons: spring, summer, autumn and winter. Data were compared with food norms and recommendations. The average caloric value of a food ration was at a correct level. The analyzed menus met the demand for nutrients to varying degrees. Below the standard was fat and protein content – in spring and summer. In spring, carbohydrate content exceeded recommended standards; the amount of protein and carbohydrates in the autumn and winter season was consistent with the current nutritional recommendations for this age group. It was also found that children diet was deficient in calcium, iron and vitamin C. Comparison of menus in terms of their compliance with the principles of rational nutrition showed that: meals were served at the right time, with proper breaks between breakfast, lunch and dinner; meals were prepared with different techniques, varied, containing seasonal vegetables and fruits. The assessment of the pre-school menu showed the need to change the composition of meals and the necessity of training for the person responsible for their composition.

138 P 85. Evaluation of the energy and nutritional value of diets used in patients on the example of a chosen hospital from Lower Silesia A. Orkusz1,2, R. Olędzki1,2, U. Kaim1,2, L. Bogacz-Radomska1,2, J. Harasym1,2, S.,Backiel2

1Adaptive Food Systems Accelerator – Science Centre, Wrocław University of Economics, Poland 2Department of Biotechnology and Food Analysis, Institute of Chemistry and Food Technology, Wrocław University of Economics, Poland Evaluation of the diet in Polish hospitals is important because it is not obligatory, and it is well known that the quality of nutrition in hospital facilities is highly unsatisfactory. The aim of this study was evaluation of energetic and nutritional value of hospital diets, administered in a chosen hospital of the Lower Silesia area based on theoretical analysis of the menus using the Dietician 2 computer program. Easily digestible diet was analyzed. The energy values, contents of protein, fat, carbohydrates, mineral ingredients: Ca, K, Mg, Fe vitamins: A, C, D, E, B6, B12 were calculated for forty hospital menus in four seasons: spring, summer, autumn and winter. Data were compared with food recommendations. In the examined hospital menus, the number of meals served and the intervals between them were correct, consistent with the principles of rational nutrition. Breakfast was served at 8:30, dinner at 12:30 and supper at 17:30. The evaluation of nutritional value of the menus demonstrated several irregularities concerning the norms. The diet was characterized by low contents of caloric value, carbohydrates, calcium, potassium, magnesium, vitamins: C and D. High dietary content of vitamin A, E, B6, B12 was found. The contents of protein and fat in menus met the obligatory standards. The planned diet was not really varied, meals were often repeated.

139 P 86. Rheological properties of ointments containing prolactin as an indicator of their quality A. Ostróżka-Cieślik1, B. Dolińska1,2, F. Ryszka2

1Department of Pharmaceutical Technology, School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia in Katowice, Sosnowiec, Poland 2“Biochefa” Pharmaceutical Research and Production Plant, Sosnowiec, Poland Prolactin (PRL, mammotropin, lactotropin) is a polypeptide hormone containing 198 amino acids and having a molecular mass of 23 kDa [1]. It has a pleiotropic effect, which indicates its great potential in designing new therapeutic methods. Dosing of PRL in the form of an ointment may be beneficial due to its low durability, limited solubility and tendency to aggregate. The aim of the study was to develop a technology for making an ointment with prolactin in order to obtain a formulation with the desired quality parameters. Ointment of emulsions-type were prepared in accordance with the requirements of Polish Pharmacopoeia XI using the Unguator mixing unit. PRL was dissolved and emulsified in amphiphilic base – Lekobaza. Rheological analysis was performed using a Lamy RM 200 Touch rheometer under isothermal conditions (T = 25±0.2°C). Viscosity tests at three shear rates 300, 700 and 1100 s-1 were carried out. The mathematical nonlinear model of Ostwald-de Wall was used for the analysis. Studies on the dependence of viscosity and tangential stress on the increasing shear rate for the obtained preparations were also performed. It has been found that ointments belong to non-Newtonian, pseudoplastic solutions with thixotropic properties. The presence of the flow limit on reograms indicates their good rheological properties. The observed change in viscosity under the influence of shear stress allows smearing ointment on the skin and good adhesion at the application site. The formulations developed may be a potential alternative to other routes of dosage of this hormone. ACKNOWLEDGEMENTS The study was supported by the SUM statutory agreement No. KNW-1-118/N/8/O „Research on the development of the drug form (solutions, ointments) with protein-peptide substances, including hormones”. REFERENCES: 1. M.E. Freeman, et al. Physiol. Rev. 80, p. 1523-631, 2000.

140 P 87. The differences in susceptibility to antiseptic agent displayed by the same Enterobacteriaceae strains grown in standard microbiological vs. artificial urine medium J. Paleczny1, M. Oleksy1, K. Dydak1, M. Wawrzyniak1, A. Junka1, M. Bartoszewicz1

1Department of Pharmaceutical Microbiology and Parasitology, Wroclaw Medical University, Wroclaw, Poland Gram-negative Enterobacteriaceae rods, due to their increasing resistance to antimicrobials and ability to form biofilm on biotic and abiotic surfaces, are common etiological factor of urinary tract infections. To compare activity of octenidine dihydrochloride (OCT) against planktonic and biofilm structures of Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae cultured in tryptic soy broth (TSB) and artificial urine medium (AUM). For the experimental purposes, strain from reference American Type Culture Collection and two clinical isolates, of whom one displayed weak and another high ability to form biofilm were applied (i.e. 3 strains of each of aforementioned species). Microbes were cultured in polystyrene microtiter plates in standard microbiology medium (TSB) or in the artificial urine medium (AUM). The used OCT concentrations range was decreasing in geometric progression, starting from 0.1%. A spectrophotometric measurement of cell density for planktonic forms was applied to assess impact of antiseptic, while in case of biofilm, level of red formazan compound synthesized by bacterial biofilm was assessed. The tested antiseptic displayed higher activity against planktonic forms grown in AUM than in TSB. The MICs (Minimal Inhibitory Concentration) were 1-2 times lower in the AUM-test compared to those performed in TSB. Also in case of biofilms, eradication level was higher in a setting which contained AUM than TSB. The conditions used for standard assay of antimicrobial activity stay in discrepancy with results obtained in environment more close to the factual one. It is also worth of noticing that it seems that the factual effectiveness of antimicrobial agents may be higher in the clinical conditions than it would be expected if only in vitro results were taken under consideration.

Research was performed by means of the scientific project no STM.D230.16.038

141 P 88. High fat and high cholesterol diet affect the level of lipopolysaccharides (LPS) and the level of proinflammatory factor – TNF-α in rat model J. Palma, D. Maciejewska , K. Skonieczna-Żydecka, N. Komorniak, J. Kikut, A. Wolska, D. Styburski, M. Skórka, W. Żwierełło, J. Kałduńska, E. Stachowska Department of Biochemistry and Human Nutrition, Pomeranian Medical University in Szczecin, Szczecin, Poland Lipopolysaccharides are molecules that are part of the external membrane of Gram-negative bacteria which colonise the human gastrointestinal tract. In circulation, LPS initiate the development of infections and act as a stimulator of immune system cells. As a response to LPS, monocytes and macrophages produce large quantities of proinflammatory cytokines, including the tumour necrosis factor alpha (TNF-α). The applied diet has an influence on the content of gut microflora, its metabolic activity and the integrity of the intestinal barrier [1-3]. We analysed the influence of a high-fat and high-cholesterol diet on LPS and TNF-α levels in the serum of Sprague-Dawley rats. The experiment was carried out on 72 male, 8-weeks-old Sprague-Dawley rats. The study group (n=36) received a high-fat and high-cholesterol diet (HFHCh) [4]. The control group (n=36) received standard food for laboratory rats (Rodent Lab Chow, PURINA). Rats from both groups were terminated after 4, 8, 12, 16 and 20 weeks of diet exposure. At each time point, 12 animals were terminated - 6 from the control group and 6 from the study group. Subsequently, blood was collected from the heart, and the animals were bled via cardiac puncture. ELISA was used to analyse LPS and TNF-α levels. LPS and TNF-α levels increased with every passing week. The results are statistically significant (p<0.05). High fat and high cholesterol diet has an influence on LPS levels in circulation and, therefore, on the increase of TNF-α concentration. REFERENCES: 1. A. J. Ahola, et al. Sci Rep. 7. “Dietary patterns reflecting healthy food choices are associated with lower serum LPS activity” 6511, 2017. 2. K. A. Kim, et al. PLoS One. 7. “High fat diet-induced gut microbiota exacerbates inflammation and obesity in mice via the TLR4 signaling pathway” e47713, 2012. 3. C. Erridge, et al. Am J Clin Nutr. 86. “A high-fat meal induces low-grade endotoxemia: evidence of a novel mechanism of postprandial inflammation” 1286-1292, 2007. 4. X.Zheng-Jie, et al. Dig Dis Sci. 55. “Characterization of High-Fat, Diet-Induced, Non-alcoholic Steatohepatitis with Fibrosis in Rats” 931-940, 2010

142 P 89. Effects of Gellan Gum based biopolymers and products of their degradation on dental stem cell condition E. Pańczyszyn1 , T. Męcik-Kronenberg2, A.M. Osyczka1 , G. Tylko1

1Department of Cell Biology and Imaging, Institute of Zoology and Biomedical Research, Faculty of Biology, Jagiellonian University, Kraków, Poland 2Department of Pathomorphology, Medical University of Silesia, Katowice, Poland Biodegradable polymers were found to be useful as carriers of cells and active substances in regenerative medicine [1]. When incorporated into inflamed and destroyed tissues, the materials slowly release therapeutic compounds and/or factors that promote differentiation of progenitor cells [1]. Since extracts from Pelargonium sidoides roots show antibacterial, anticancer and antiinflammatory activities [2], a root extract (PSREs) and isolated fraction of proanthocyanidins (PCANs) were mixed with Gellan Gum (GG) crossed-linked in the presence of EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) or CaCl2 to obtain three-dimensional structure of hydrogels. Cytotoxicity of the biocompounds were tested in vitro on both Dental Pulp Stem Cells (DPSCs) and Periodontal Ligament Stem Cells (PDLSCs). The cells were cultured in 24-well plates in Serum Containing Medium (SCM) till 70-80% confluency and then treated for 24h with SCM media derived from pure biopolymers (GG), those loaded with P. sidoides active substances (PSREs/PCANs), and fresh SCM (Control). Cell viability/proliferation was tested by MTS assay and the results indicate that GG biopolymers seem to be non-toxic for both types of cells, whereas presence of GG loaded with PSREs/PCANs significantly decreased DP/PDLSCs viability/proliferation. Moreover, cell treatment with GG or products of its degradation crosslinked with EDC stimulate both types of cells to formation of intracellular vesicles. The studies reveal that Gellan Gum-based biopolymers/hydrogels can be successfully used in regeneration of periodontal tissues. Additionally, such biocompounds loaded with plant-derived active substances might inhibit microbial growth and inflammation of gum and tooth tissues. However, determination of a process of vesicle formation in DP/PDLSCs requires further investigations. ACKNOWLEDGEMENTS The authors thank to M.ERA-NET2 grant PELARGODONT (NCN 2016/22/Z/ST5/00692) for the financial support REFERENCES: 1. C. Martins et al. Adv. Healthcare Mater. p. 1701035, 2017. 2. N. Savickiene et al. Materials 11, p. 1499, 2018.

143 P 90. Design, synthesis, spectroscopic and in silico studies of new Mannich base derivatives of pyrrolo[3,4-d]pyridazinone as potential analgesic agents K. Peregrym1, K. Potyrak1, P. Zając1, A. Zborowska1, Ł. Szczukowski2

1 Student Scientific Club of Chemistry of Drugs, Wrocław Medical University, Borowska 211, 50-556 Wrocław, Poland 2Department of Chemistry of Drugs, Wrocław Medical University, Borowska 211, 50-556 Wrocław, Poland Derivatives of pyrrolo[3,4-d]pyridazinone present a wide range of biological activities e.g. antibacterial, anticancer or analgesic. When considering analgesic activity, some of them were even stronger than morphine [1, 2]. This fact was a powerful prompt to design and synthesise new analgesic and anti-inflammatory compounds, bearing pyrrolo[3,4-d]pyridazinone scaffold, which would be deprived of adverse effects characteristic for NSAIDs, especially gastrotoxicity. We report herein the synthesis of two novel Mannich base derivatives of pyrrolo [3,4-d]pyridazinone with predicted antinociceptive activity. In order to reduce ulcerogenicity we turned carboxyl group into hydrazide and, in the next step, obtained the ring of 1,3,4-oxadiazole-2-thione. Such modification, performed also on commonly used NSAIDs, e.g. Ibuprofenum or Diclofenacum, allows to receive compounds possessing promising analgesic and anti-inflammatory activity, but devoid of gastrointestinal toxicity [3-5]. In the last step of synthesis, derivatives of pyrrolo[3,4-d]pyridazinone were condensed with formaldehyde and secondary amines, in this case with 4-substituted piperazine, to receive final Mannich bases. Structures of new compounds were confirmed by spectroscopic techniques: MS, FTIR, 1H NMR, 13C NMR and element analysis. We have also performed some in silico studies in order to estimate potential pharmacological activity and bioavailability of received derivatives. Our own investigations confirmed that both obtained Mannich bases could possess good analgesic activity and satisfactory bioavailability. According to literature, presence of the ring of 1,3,4-oxadiazole-2-thione and 4-substituted piperazine moiety could be essential for pharmacological profile and low toxicity of new compounds as well. REFERENCES: 1. Malinka W. et. al. Zgłoszenie patentowe P 3869668 2008. 2. Malinka W. et al. Eur. J. Med. Chem. 46 p. 4992-4999 2011. 3. Koksal M. et. al. Arch. Pharm. Chem. Life Sci. 350 1700153 2017. 4. Palkar M.B. et. al. Bioorganic & Med. Chem 22 p. 2855-2866 2014. 5. Manjunatha K. et al. Eur. J. Med. Chem.45 p. 5225-5233 2010.

144 P 91. Cooperation is the key to success – synergistic combinations of essential oils E. Piątkowska1, M. Brożyna2, A. Starzec3, F. Kominek4

1Department of Pharmaceutical Microbiology and Parasitology, Wroclaw Medical University, Wrocław, Poland 2Department of Pharmaceutical Microbiology and Parasitology, Wroclaw Medical University, Wroclaw, Poland 3Department of Pharmacognosy and Herbal Medicines, Wroclaw Medical University, Wroclaw, Poland 4Department of Pharmacognosy and Herbal Medicines, Wroclaw Medical University, Wroclaw, Poland Due to the development of microorganisms' resistance to various antibiotics, alternative methods of treating bacteria and fungi infections need to be found. As a novel solution of this problem, essential oils (EOs) that have been proved to possess strong antimicrobial activity are proposed. EOs are complex mixtures of numerous constituents, hence their antimicrobial properties are usually the result of combined action of several compounds which can work synergistically. Moreover, different essential oils in combinations may exert synergistic interactions and significantly reduce MIC (minimum inhibitory concentration) values against various microorganisms. Taking advantage of their synergistic effects, it is suggested that they could be used to enhance microorganisms' susceptibility to most groups of antibiotics. Thus, essential oils are promising agents which not only can be used as antibiotic adjuvants but also as preservatives in cosmetics and food.The aim of this review was to analyse synergistic effects of selected essential oils (e.g. thyme oil, basil oil, lemon oil) in combinations with themselves as well as with antibiotics in order to find the most relevant ones for pharmaceutical industry.

145 P 92. The main cannabinoid receptor expression in healthy mammal tissue P. Przybycień

Jagiellonian University, Medical College It is well-known that the endocannabinoid system (ECS) is a natural system composed of endocannabinoids and their receptors. It is spread throughout the mammalian central nervous system and peripheral organs. The major ligand of the CB1-receptor is AEA – anandamide. This system is involved in many processes of the mammalian body, such as pain, sensation, mood, appetite, fertility or pregnancy. The aim of the study was to assess the CB1-R levels in various parts of the CNS and female reproductive system. The first stage of the study was organ extraction from lab mice. Subsequently, each sample was homogenized and centrifuged. We measured the total protein concentration and performed Western Blot assays on the extracts. Finally, the receptors were visualized using immunochemiluminescence. A detailed comparison of the results in tested tissues was performed. The study focused on two types of samples of CNS tissue and two types of female reproductive tissue. A higher concentration of the receptor was noted in the encephalon compared to the cerebellum and in uterine tissue compared to the oviduct. Receptor concentration in the brain and uterus was similar. In this study we have confirmed a variable concentration of the CB-1 receptor in murine tissue. The data, along with available literature, suggests that the endocannabinoid system is involved in regulation of physiological function. There may be a need to examine other cannabinoid receptors, particularly those discovered recently, in similar tissues.

146 P 93. Acute leukemia in people with trisomy 21 chromosome A. Czyż1, M. Król1, O. Przybyszewski1, A. Radajewska1 Supervisors: E. Barg2, H. Moreira2, B. Wiatrak2

1Student Research Group of Flow Cytometry and Biomedical Research, Wroclaw Medical University 2Department of Basic Medical Sciences, Wroclaw Medical University Transient abnormal myelopoeiasis (TAM) is a unique haematological disorder that occurs in 5-10% of new-borns with Down Syndrome (DS). TAM is considered a self-limiting disease that occurred within the first few months after birth. However, about 20% of patients with TAM die during infancy due to acute leukemia or liver failure. It has long been recognized that children with trisomy 21 chromosome (DS) have a clearly increased risk of acute leukaemia, both acute megakaryocytic leukemia (M7 FAB) – myeloid (AML) and type B acute lymphoblastic leukemia (B-ALL). The unique characteristics of DS-associated leukemia not only indicate the key role of trisomy 21 in the pathogenesis of chromosome, but may allow the study to know completely new mechanisms of inducing leukemia at the embryonic level. This means that trisomy 21 alters the functioning of fetal stem cells, including the stem cell of haematopoiesis (HSC), causing numerous abnormalities in myelopoesis and lymphopoiesis. In addition, platelet-derived growth factor (PDGF) and transforming growth factor (TGF -β1) were found to be strongly expressed on megakaryoblasts, which may explain liver fibrosis in patients with DS with TAM, and studies have also shown that the liver deteriorates even after hematologic remission. The pathogenesis of TAM and acute leukemia in people with Down syndrome is probably very complex, but indicates the significant role of chromosome 21 in the mechanism of disease formation and progression. Analysis of changes taking place in stem cells may bring closer the phenomenon of liver fibrosis or neoplastic cell transformation preceded by TAM. The presented work is the result of a review of the latest scientific literature on acute leukemia and their course in people with trisomy 21 chromosome.

147 P 94. Electrochemotheraputic approach in human breast cancer cells with drug resistance N. Rembiałkowska, J. Saczko, J. Kulbacka

Department of Molecular and Cellular Biology, Wroclaw Medical University, Borowska 211 A St., 50-556 Wroclaw, Poland; Resistance of cancer cells to chemotherapy is one of the main causes of an ineffectiveness of the current pharmacotherapy. Cancer cells are able to modify the activity of numerous proteins involved in transport processes, xenobiotics metabolism or DNA repair. Until now, numerous attempts to reduce the scale of multidrug resistance (MDR) have been unsuccessful to the extent enabling effective systemic therapy of neoplastic diseases. Therefore, further research is needed on the methods of inhibiting MDR tumor-determining proteins or new methods such as electrochemotherapy. The aim of the study the expression of MDR proteins was evaluated: P-gp and BCRP, involving inhibitors as Elacridar and Fumitremorgin C after chemotherapy and electrochemotherapy. Here we investigated human breast adenocarcinoma cells of wild type (MCF-7/WT) and doxorubicin resistant cells (MCF-7/DOX). The ESOPE (European Standard Operating Procedure on Electrochemotherapy) electroporation parameters were applied: 1000 V/cm, 100 µs, 8 pulses. In our experiments a low-conductive medium containing phosphate buffer with sucrose was used. The expression of drug resistance proteins after the applied treatment was evaluated by immunocytochemical assay. Our results indicate the most pronounced effect of ECT (electrochemotherapy) combined with MDR modulators was observed in resistant cells and significant decrease of P-gp expression. Sensitive cells occurred less susceptible to applied therapy. ACKNOWLEDGEMENTS This research was supported by National Science Center, Poland, grant No. DEC- 2015/19/N/NZ7/01105, (PI: N. Rembiałkowska).

148 P 95. Design and molecular docking study of novel colchicine derivatives as a potential tubulin polymerization inhibitors K. Rogalski1, A. Ryglowska1, K. Lizoń1, Ż. Czyżnikowska2

1Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, 50-556 Wrocław, Poland 2Department of Inorganic Chemistry, Wroclaw Medical University, 50-556 Wrocław, Poland Heterodimers of tubulin play an important role in growth and cell division. They can be also involved in intracellular transport and morphogenesis [1]. Colchicine is first well known compound which can bind to the tubulin what disturb its organization and inhibit microtubule formation. It is responsible not only for antimitotic effect on cells but also arrests cells during interphase. Therefore the colchicine binding site is the most promising target for anticancer agents [2]. In the present study new colchicine derivatives were designed as a inhibitors of tubulin polymerization. In order to determine the possible binding modes of proposed compounds docking studies were carried out. According to the results the analyzed derivatives can interact with the active site of tubulin and amino acid residues responsible for colchicine binding. Similar manner of interactions and obtained values of inhibition constant obtained for investigated systems suggested further studies in order to confirm their biological activity. REFERENCES: 1. S.B. Hastie, Pharmacol Ther. vol. 51 pp. 377-401, 1991. 2. L. Yan et. al Pharm Res. vol. 29 pp. 2943-2971, 2012.

149 P 96. Biotechnological method of enrichment of natural phosphatidylcholine with 3,4-dimethoxycinnamic acid M. Rychlicka1, K. Puchrowicz1, N. Niezgoda1, A. Gliszczyńska1

1Department of Chemistry, Wroclaw University of Environmental and Life Science, Wroclaw, Poland Phenolic acids, especially cinnamic acid derivatives, are a widespread group of non-nutritive compounds found in foods with strong antioxidant potential and resulting from this other pro- health properties like anticancer, antidiabetic, antimicrobial or anti-inflammatory activity [1]. Therefore, phenolic acids are a valuable group of natural compounds for the food industry, which can act as health-safe natural food preservatives and nutraceuticals helpful in preventing the development of many civilization diseases. The practical use of cinnamic acid derivatives in the industry is unfortunately hampered due to their poor solubility in a hydrophilic and hydrophobic environment and low bioavailability in the body. The latest research is therefore focused on improving their physical and chemical properties, which would allow for their practical application in the industry. Promising in this respect are modifications of the structure of cinnamic acid derivatives via covalent bonding them with a lipid carrier, especially with phosphatidylcholine, which possesses amphiphilic character, is the source of unsaturated fatty acids (position sn-2) and choline (position sn- 3) and is characterized by high bioavailability in the human body. Presented research conclude the development and optimization of a one-stage, biotechnological method of enriching natural phosphatidylcholine isolated from egg yolk into 3,4-dimetho- xycinnamic, in order to extend their use in the food industry as safe for health biomolecules that can act as food preservatives and nutraceuticals with health-promoting effect. REFERENCES: 1. El-Seedi HR et al, J Agric Food Chem, 60: 10877-10895, 2012.

150 P 97. Rheological properties of the hydrogels containing liposomal vitamin C B. Sarecka-Hujar, H. Józefiak, B. Dolińska

Department of Pharmaceutical Technology, Medical University of Silesia in Katowice, Poland Semi-solid formulations for topical use are one of the most common pharmaceutical preparations. From the physicochemical point of view, they are systems with plastic properties of gels and belong to the category of non-Newtonian bodies. They may contain one or more drug substances which are dissolved, emulsified or suspended in the medium. The composition of the ointment base influence the speed and depth of the active substance penetration through the skin. The proper consistency of the ointment base is reflected in rheological properties, which include among others flow curve and spreadability (plastic viscosity) as well as adhesion to the skin or mucous membranes. The aim of the present study was to assess rheological properties of two types of hypromellose hydrogels (5% and 10%) containing liposomal vitamin C in a concentrations of 20%. Viscosity of the obtained formulations depending on shear rate (D=300 s-1, D=800 s-1 and D=2400 s-1) at 23°C was performed in a Reometer 200 Touch (Lamy). In the analysis we used a DIN-33 measuring set, consisting of a spindle and an application tube, in which 5 ml of each of the prepared hydrogels was applied. We observed slightly increased viscosity of hydrogel with 5% hypromellose with increasing shear rate while for 10% hypromellose gel – the reverse trend was found.

151 P 98. Prevalence of Cryptosporidium spp. in renal transplant recipients J. Sell1, Ż. Kopacz1, K. Jakuszko2, M. Kicia1

1Department of Biology and Medical Parasitology, Wroclaw Medical University, Wroclaw, Poland; 2Department of Nephrology and Transplantation Medicine, Wroclaw Medical University, Wroclaw, Poland; Organ transplant recipients represent a group of patients highly susceptible to opportunistic infections due to lifelong immunosuppressive therapy. One of the most ubiquitous enteric protozoa pathogen recognised as a cause of diarrhea in humans is Cryptosporidium spp [1]. Faecal-oral route is the main way of transmission of infection, thus people become infected by ingestion of food or water contaminated with oocysts or, less frequently, by inhalation of oocysts. Therefore, parasite develops in digestive system and/or respiratory system. Infections can vary from asymptomatic form to acute gastroenteritis. Lack of routine laboratory testing in risk groups causes that infections of Cryptosporidium may be highly underdiagnosed, posing important epidemiological problem. The aim of our study was to determine the prevalence of Cryptosporidium spp. infections among renal transplant recipients. A total of 148 samples, sputum (77/148) and stool (71/148), were obtained from patients being under the care of Department of Nephrology and Transplantation Medicine, Wroclaw Medical University (Wroclaw, Poland). Samples were screened by using genus-specific nested-PCR protocols amplifying SSU rRNA gene sequences of Cryptosporidium spp. followed by genotyping. Nested-PCR showed Cryptosporidium DNA presence in stool samples obtained from 5 patients. However, detailed sequencing and phylogenetic analysis is needed to confirm the species/genotype. No cryptosporidial infection was confirmed in sputum samples. Our results indicate the risk of cryptosporidial infection in digestive tract of renal transplant recipients. REFERENCES: 1. Cryptosporidium infection in solid organ transplantation, World Journal of Transplantation vol. 6(issue3), September 24, 2016.

152 P 99. Bacterial microflora isolated from wounds of patients with diabetic foot infection hospitalized in the general surgery ward M. Sępek 1, Z. Sycz2, J. Jeżewska3, J. Rudnicki1

1 Department of General, Minimally Invasive and Endocrine Surgery, Wrocław Medical University, Borowska 213, 50-556 Wrocław, Poland 2 Department of Biology and Medical Parasitology, Wrocław Medical University, Mikulicza-Radeckiego 9, 50-367 Wrocław, Poland 3 Department of Public Health, Division of Medical Social Sciences, Wrocław Medical University, Bartla 5, 51-618 Wrocław, Poland Skin lesions are common to every people during their lifespan with or without care required. Unfortunately for diabetic people every scratch can become a seriously problem, i.e. in the form of non-healing infected wound. Additionally, postponed wound healing can be caused by presence of bacteria especially in polymicrobial infection of ulcers. Particular research interest exists in the relationship between skin microflora and diabetic chronic wounds. The main aim of this study was to establish a bacterial microflora existing in chronic ulcerations in people with diabetic foot hospitalized in the general surgery ward in years from 2013 to 2019. Swab specimens of chronic foot ulcerations were obtained using the Levine technique. The VITEK 2 system (BioMérieux) for rapid identification of clinical bacterial strains was used. The overall composition of the diabetic wound bacterial species was dominance of Staphylococcus aureus, as well as Enteroccocus faecalis, Streptococcus spp., Escherichia coli, Pseudomonas aeruginosa were found. Some bacterial species, such as Enterococcus faecium, Enterobacter cloacae, Staphylococcus hominis MRS, Bacteroides fragilis, Anaerococcus prevotti, Lactobacillus fermentum were specific to individuals.

153 P 100. Submicron resolution imaging of biological samples supported with plasma etching and precise positioning system A. Sikora1, P. Żurek2, M. Moczała1, M. Karpińska2, M. Poznar3, P. Dobryszycki3

1Electrotechnical Institute, Division of Electrotechnology and Materials Science, M. Skłodowskiej-Curie 55/61, 50-369 Wrocław, Poland 2Active Students Association, Association of Polish Electrical Engineers SEP (Branch No. 1 in Wrocław), M. Skłodowskiej-Curie 55/61, 50-369 Wrocław, Poland 3Wrocław University of Science and Technology, Faculty of Chemistry, Department of Biochemistry, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland Development of advanced diagnostic techniques and tools allows to perform more and more complicated investigation procedures. In particular the diversity and complexity of biological samples requires enabling a wide range of research solutions providing desired information about their structure and various properties [1-6]. Recently a plasma etching system was successfully tested in terms of imaging of the structure of investigated objects. It allowed to perform iterative imaging/ etching procedure, providing the access to the inner structures of the samples and quasi-3D tomography. Successful and effective investigation of certain area of the sample using single instrument in repetitive fashion or the set of various diagnostic techniques requires access to that same spot at every stage. It can be difficult concerning the submicron scale of surface imaging related to milimeter or centimetre-scale samples. Therefore universal and reliable tool providing precise and easy sample positioning is necessary. In presented research, also such solution was successfully tested. This positioning tool enabled easy and effective correlative imaging, therefore the research quality was improved as well as the operation time was reduced. Its versatility allows to perform the imaging using a variety of methods such as: optical microscopy based techniques, scanning electron microscopy, Raman imaging, atomic force microscopy, nanoindentation and others. A set of research results will be presented, revealing the application range of both: plasma etching and correlative imaging solutions. Those tools made possible a unique, repetitive observation of certain area of the sample, while certain kind of medium was applied. Also, a wide range of diagnostic methods could be used with desired spatial precision. ACKNOWLEDGEMENTS The authors thank Electrotechnical Institute for providing the lab facility. REFERENCES: 1. M. Moczała, et al. Materials Science – Poland 36(1), 75-79, 2018. 2. B. Hrycak, et al. IEEE Transactions on Plasma Science, in print, 2019. 3. J. Kulbacka, et al. 15th International Bioelectrics Symposium, Book of Abstracts, p. 34, 2018. 4. A. Sikora, Meas. Sci. Technol. 25, 55401, 2014. 5. A. Sikora, Nanoscience and Nanometrology, 3(1), 6-11, 2017. 6. M. Dykas, Correscopy Company materials, 01/2017, 2017.

154 P 101. Accumulation of lead (Pb) in THP1 cells depending on the applied concentration D. Simińska1, E. Metryka1, P. Kupnicka1, P. Kapczuk1, D. Styburski2, M. Bosiacki3

1Department of Biochemistry, Pomeranian Medical University in Szczecin, Powstańców Wlkp. 72 St, 70-111 Szczecin 2 Department of Biochemistry and Human Nutrition, Pomeranian Medical University in Szczecin, Broniewskiego 24 St, 71-460 Szczecin 3Department of Medical Rehabilitation and Clinical Physiotherapy, Pomeranian Medical University in Szczecin, Żołnierska 54 St, 71-210 Szczecin Despite the introduction of various preventive measures lead (Pb) is a heavy metal widely distributed in nature, which in the environment is not biodegradable or breaks down and accumulates in the tissues of living organisms. This element is primarily absorbed through the respiratory tract or digestive system and with small extent through the skin. Chronic exposure to even low concentration of this metal can be harmful, because currently it is considered that there is no threshold level for lead toxicity and that the metal can adversely affect even very low concentrations in the blood. The constant presence of Pb compounds makes it necessary to study their effect on the functioning of organisms. The aim of the study was to assess the accumulation of Pb in macrophages of the THP-1 cell line. The studies were performed on THP-1 cells. The cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 IU/ml penicillin and 10 μg/ml streptomycin at 37°C, 5% CO2 and 95% humidity. PMA was used to differentiate monocytes to macrophages. Cells were subjected to 24h and 48h incubation with lead acetate, with a final concentration of Pb: 1.25μg/dL; 2.5μg/dL; 5μg/dL; 10μg/dL. Control groups were cultured without the irritant or with the addition of sodium acetate at a concentration of 10μg/dL. The Pb concentration in cells and medium was determined using the ICP-OES method. The range of Pb accumulation level in cells was 11-32%. Comparing incubation period, Pb more strongly accumulated in cells after 48 hours. This increase was significant in cells exposed to 2.5μg/dL; 5μg/dL; 10μg/dL Pb comparing to 24h incubation. Evaluating cells after 24h there was a greater accumulation of the analyzed element in cells grown with a Pb solution at a concentration of 5μg/dL in relation to cells exposed to 1.25μg/dL and 2.5μg/dL Pb and in relation to both control groups. 48h incubation resulted in a significant increase in the accumulation of Pb in cells with 10μg/dL Pb vs groups with 5μg/dL Pb and 1.25μg/dL Pb and vs pure control. The increase in Pb in cells with 5μg/dL Pb and with 2.5μg/dL Pb was significant vs the group with 1.25μg/dL Pb and both controls. Pb accumulates in macrophages of the THP-1 cell line. The concentration of Pb added to the culture medium influences the degree of accumulation of the element in the cells. REFERENCES: 1. Skoczyńska, A., Gruber, K., Belowska Bień, K., & Mlynek, V. 2007. “Ryzyko chorób układu krążenia u pracowników huty szkła narażonych na działanie ołowiu” Medycyna Pracy. 2007, strony 475-483. 2. Nazimek K. i Bryniarski K. 2012. “Aktywność biologiczna makrofagów w zdrowiu” Postępy Higieny I Medycyny Doświadczalnej (online). 2012, strony 507-520. 3. Qin, Z. 2012. “The use of THP-1 cells as a model for mimicking the function and regulation of monocytes and macrophages in the vasculature” Atherosclerosis. 2012, strony 2-11.

155 P 102. The effect of metal ions binding on the structure of Nucleobindin-2 from Gallus gallus and Homo sapiens A. Skorupska, A. Ożyhar, D. Bystranowska

Department of Biochemistry, Faculty of Chemistry, Wrocław University of Science and Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland Nucleobindin-2 (Nucb2) is a multidomain protein, which consists of two EF-hand domains [1]. EF-hand domains are responsible for calcium and magnesium ions binding [2]. Nucb2 is a protein involved in multiple biological processes i.e. biomineralization [3], food intake inhibition [4] and cancerogenesis [5]. Nucb2 is also implicated in a wide range of cellular interactions, which seems to be calcium-dependent [6]. The aim of our experiments was to focus on the effect of calcium and magnesium ions on the structure of Nucb2 molecules. We utilized two homologs of Nucb2 from Gallus gallus and Homo sapiens, which share 78% amino acid sequence identity to obtain broader and more representative set of results. We performed two techniques: circular dichroism (CD) and limited proteolysis to elucidate the magnesium and calcium ion effect. The CD spectra showed that both proteins undergo disorder-to-order transition upon calcium ion binding. Additionally, the structure of Nucb2s in the presence of CaCl2 is more resistant to proteolysis. Magnesium ions have a slightly protective effect on hsNucb2 structure. Interestingly, ggNucb2 is not affected by magnesium ions. REFERENCES: 1. S. Barnikol-Watanabe, et al. Biol. Chem. Hoppe Seyler, 375: 497-512, 1994. 2. A. Malmendal, et al. Biochemistry, 38:11844-11850, 1999. 3. M. Rose-Martel, et al. J Proteomics, 116:81-96, 2015. 4. S. Oh-I, et al. Nature. Vol. 12, 200. 5. A. Skorupska, et al. Postępy Higieny i Medycyny Doświadczalnej, 72: 1084-1096, 2018. 6. N. Taniguchi, et al. J. Biol. Chem, 275: 31674-31681, 2000.

156 P 103. Does exposure to fluorine affect COX activity in the rats brain? M. Skórka-Majewicz1, W. Żwierełło1, D. Styburski1, K. Dec1, J. Kałduńska1, J. Kikut1, N. Komorniak1, A. Wolska1, J. Palma1, D. Maciejewska1, P. Kupnicka2, E. Metryka2

1 The Department of Biochemistry and Human Nutrition, Pomeranian Medical University 2 The Department of Biochemistry, Pomeranian Medical University The constant exposure to increased concentrations of fluorine may lead to chronic intoxication with this element, which results in numerous negative health consequences, including activation of COX enzymes responsible for the formation of inflammation. Determine whether exposure to fluoride during the development affects cyclooxygenases activity and the synthesis of prostanoids in different brain structures. The pregnant females Wistar rats was supplied with water including a sodium fluoride solution with the concentration of 50 mg/L (ad libitum). 12 pups rats (6 tested and 6 controls) were included in the study. The process was continued from the moment the pups are born to the time they are maturity. The time of observation after exposure to fluoride solutions began in the prenatal period to the end of the 3rd month of life. After this time, the rats was terminated and their organs and sera was collected for further research. Applied methods:  Potentiometry – analysis of fluoride concentration;  qRT-PCR – analysis of COX1 and COX2 expression;  ELISA – analysis PGE2 and TXB2 concentrations;  Immunohistochemistry – COX1 and COX2 proteins immunoexpression; The analysis of the expression of COX1 and COX2 showed that long-term exposure to fluoride during prenatal and postnatal development significantly influenced the expression of these genes of the studied enzymes. The cerebellum, hippocampus and striatum of those from the fluoride exposed group showed significantly lower expressions of both COX1 and COX2, whereas in the prefrontal cortex only COX1 expression was significantly lower.

157 P 104. Sirtuin1 as a marker of oxidative stress in women with PCOS A. Słowiak1, A. Bizoń2

1Students Scientific Society at the Department of Biomedical Environmental Analysis, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, Wrocław, Poland 2Department of Biomedical and Environmental Analysis, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, Wrocław, Poland Sirtuin1 (SIRT1) is a NAD+-dependent deacetylase regulating a variety of signaling pathways involved in metabolism, inflammation, cellular senescence, proliferation, apoptosis, and DNA damage response, due to its ability to deacetylate a multiplicity of developmental factors [1]. Also, it has been proved that SIRT1 protects against oxidative stress (OS) by up-regulating FOXO3-dependent antioxidant genes [2]. The OS is intimately involved in pathological processes which accompany in polycystic ovary syndrome (PCOS) [3,4]. OS has been demonstrated to be associated with obesity, insulin resistance, inflammation, and hyperandrogenemia, which are the common characteristics and potential inducers of PCOS [5]. Previous studies indicate that the expression of sirtuin1 is reduced in case of obesity and insulin resistance [6]. Also, it has been shown that sirtuin1 is expressed in the ovarian follicular cells [7] where the insulin and insulin-like growth factors are important in steroid hormone synthesis and ovarian follicular development [8]. SIRT1 can promote insulin secretion and increase its impact by adjusting the interactions between the insulin-related proteins and insulin signal transduction pathway [9]. Therefore, an abnormal insulin signal transduction can also exist locally in the PCOS, leading to an increased androgen synthesis in the ovaries, which disturbs ovulation [8]. OS is intimately involved in pathological processes which accompany in PCOS including hormone disorders, insulin resistance, hyperandrogenism, and obesity. The sirtuin1 could participate in these disorders and can be used as their potential marker. REFERENCES: 1. Finkel T., et al. “Recent progress in the biology and physiology of sirtuins” Nature. 460:587-91. 2009. 2. Alcendor RR., et al. ”Sirt1 regulates aging and resistance to oxidative stress in the heart” Circ Res. 100:1512-21. 2007. 3. Murri M., et al. “Circulating markers of oxidative stress and polycystic ovary syndrome (PCOS): a systematic review and meta-analysis” Human Reproduction Update. 19(3):268-288. 2013. 4. Lee J. Y., et al. “Role of oxidative stress in polycystic ovary syndrome” Current Women's Health Reviews 6(2):96-107. 2010. 5. Tao Z., et al. “Roles of Oxidative Stress in Polycystic Ovary Syndrome and Cancers” Oxid Med Cell Longev. vol. 2016:14 pages. 2016. 6. Stefanowicz M., et al. “Adipose tissue, but not skeletal muscle, sirtuin1 expression is decreased in obesity and related to insulin sensitivity Endocrine” 60(2):263-271. 2018. 7. Yoshihiro M., et al.” Resveratrol promotes expression of SIRT1 and StAR in rat ovarian granulosa cells: an implicative role of SIRT1 in the ovary” Reprod Biol Endocrinol 10:14. 2012. 8. Sonntag B., et al. “Metformin alters insulin signaling and viability of human granulosa cells” Fertil Steril. 84:1173-1179. 2005. 9. Sun C., et al. “SIRT1 improves insulin sensitivity under insulin-resistant conditions by repressing PTP1B” Cell Metab 6:307-19. 2007.

158 P 105. Hydrogels as potential forms for Central Nervous System drug delivery M. Smoleński1, K. Małolepsza-Jarmołowska1

1Department of Drugs Form Technology, Wroclaw Medical University, Wrocław, Poland Central Nervous System (CNS) diseases, disorders and injuries are one of the major challenges for medicine and pharmacy. Blood-brain barrier and low brain permeability, which limit efficiency of drug therapy, often requires usage of high dosage of medicaments and lead to poor quality of life due to possible adverse effects. Nanocarriers and alternative route of administration are possible ways to overcome this issue. Hydrogels are highly adjustable and can change their properties in different environment which might be used to design new drug forms or drug carriers. The aim of this study was to analyse recent reports about hydrogels as potential material for CNS drug delivery. Alginate, chitosan, collagen I, fibrin, hyaluronic acid, methylcellulose and specific types of peptides are potentially applicable materials for hydrogels preparation and might be useful in CNS injuries treatment [1]. Self-assembling peptide nanofiber hydrogels show similar structure to CNS extracellular matrix which allows to create scaffold for axonal growth or environment for encapsulated cells [2]. There are several preclinical in vivo studies on intranasal route of anti-Alzheimer drugs administration. Many of them are based on mucoadhesive hydrogels containing therapeutic particles to overcome mucociliary clearance and usage of high drug dosage [3,4]. Thermoreversible nasal gel – which included ropinrole as active agent, Pluronic F- 127 and hydroxylpropyl methylcellulose – was tested as non-invasive drug for Parkinson’s disease. In vivo studies on mice showed five times higher bioavability of ropinrole intranasally administrated than after intravenous injection [5]. Further studies on potential use of hydrogels in CNS disfunction are needed. REFERENCES: 1. V.A. Kornev, et al. Comput Struct Biotec vol.16 p. 488-502, 2018. 2. X. Liu, et al. Front. Mater. Sci. vol. 9(1) p. 1-13,2015. 3. A. Mukta, et al. J Control Release vol. 281 p. 139-177, 2018. 4. B.A. Aderibigbe. Pharmaceutics vol. 10, 2018. 5. M. Rao, et al. Drug Dev Ind Pharm vol. 43 p. 142-150, 2017.

159 P 106. The effect of O-glycosylation on cyanidin interaction with HMEC-1 cells – structure-activity relationships K. Solarska-Ściuk1, S. Cyboran-Mikołajczyk1, K. Mieszała2, N. Glatzel-Plucińska2, H. Kleszczyńska1 1Department of Physics and Biophysics, Wrocław University of Environmental and Life Sciences, Wrocław, Poland 2Department of Histology and Embryology, Medical University of Wrocław, Wrocław, Poland Anthocyanins are the most commonly occurring pigments in fruits, vegetables and flowers. This study present the effect of cyanidin and its O-glycosides on human dermal vascular endothelial cells (HMEC-1).The main goal was determined the biological activities of cyanidin (C), cyanidin-3-0-arabinoside (CA), cyanidin-3-0-glucoside (CG), cyanidin-3-0-galactoside (CGA), cyanidin-3-0-rutinoside (CR) and cyanidin-3-5-diglucoside (CDG) with cells. The compounds’ cytotoxic activity, ability to induce apoptosis, cell cycle and intracellular reactive oxygen species (ROS) generation, as well as inhibitory activity against AAPH-inducted oxidative stress, were determined in relation to cells. We demonstrated that biological activity of cyanidin and its O-glycosides strongly depends on the number and type of sugar substituents. The compounds used are practically non-cytotoxic, and do not induce apoptosis or disturb the progression of the cell cycle. Their presence in the cell membrane affects intracellular reactive oxygen species generation under physiological and AAPH-induced oxidative stress conditions. C, CA, CG and CGA reduced the intracellular ROS generation more than CR and CDG. The results suggest a potential beneficial effect of presented compounds on the cardiovascular system. They also may prove helpful in designing dietary supplements and medicines based on natural substances that show protective and/or therapeutic effects in pathological conditions associated with excess of free radicals. ACKNOWLEDGEMENTS This work was supported by grant no. DEC-2017/01/X/NZ9/00908 from the National Science Centre, Poland and from funds of the statutory activities of the Department of Physics and Biophysics, Wrocław University of Environmental and Life Sciences, grants no. B010/0028/18.

160 P 107. Immunoglobulin G glycosylation in endometriosis K. Sołkiewicz, E. M. Kratz

Department of Laboratory Diagnostics, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, Wroclaw, Poland Endometriosis is defined as the extrauterine growth of endometrial glandular epithelial and stromal cells, generally found over pelvic peritoneal and visceral surfaces. It affects about 10% of women in the reproductive age and it is one of the most common causes of infertility [1]. The concentrations of immunoglobulin G and A antibodies (IgG and IgA, respectively) are reported to be increased in some women with endometriosis [2]. Immunoglobulins are glycoproteins produced by B cells that play a crucial role in protective immunity [3]. Immunoglobulin G is the most abundant glycoprotein in human serum [4]. Based on the amino acid sequence of the constant regions of the heavy chains, IgGs can be divided into four subclasses IgG 1-4 [3]. All subclasses have a single-conserved N-linked glycosylation site at Asn297 of the heavy chain. 10–30% of IgGs are N-glycosylated, mainly in Fc region, and they are sialylated and fucosylated biantennary oligosaccharide structures. Glycosylation plays a key role in antibody function, and IgG N-glycans are essential for the proper activity of the immune system [4]. Fc glycans are important for IgG effector functions, whereas Fab oligosaccharides modulate antigen binding. The most variable residue of the Fc sugar core is the galactose. Low-galactose-containing IgG glycoforms are observed in patients with various chronic inflammatory and infectious diseases [5]. Changes in glycosylation were also observed in women with advanced endometriosis in comparison to the group of healthy subjects [1]. REFERENCES: 1. Eutopic Endometrium From Women With Endometriosis Shows Altered Ultrastructure and Glycosylation Compared to That From Healthy Controls-A Pilot Observational Study, C.J. Jones, I.M. Inuwa, L.G. Nardo, P. Litta, A.T. Fazleabas, Reprod Sci., 16(6), 559-572, 2009. 2. The Practice Committee of the American Society for Reproductive Medicine (ASRM): Endometriosis and infertility: a committee opinion. Fertil Steril., 98,591-598, 2012. 3. Human plasma protein N-glycosylation F. Clerc, K.R. Reiding, B.C. Jansen, G.S. M. Kammeijer, A. Bondt, M. Wuhrer, Glycoconj J 33, 309-343, 2016. 4. IgG glycosylation in autoimmune diseases K. Kozłowska, M. Rydlewska, M. Ząbczyńska, Ewa Pocheć Postepy Hig Med Dosw 72, 975-990, 2018. 5. Impact of Glycosylation on Effector Functions of Therapeutic IgG, R. Abès, J.L. Teillaud, Pharmaceuticals 3, 146-157, 2010.

161 P 108. Expression of PD-L1 in B-cell non-Hodgkin lymphomas J. Spętana

Department of Pathomorphology and Oncological Cytology, Wroclaw Medical University, Wroclaw, Poland Numerous scientific studies have proved that some cancers have the ability to avoid the body's immune response through various mechanisms. One of them is the activation of inhibitory receptors on T lymphocytes, which are key cells capable of recognizing and killing cancer cells. The activity of T lymphocytes is strictly regulated by activating inhibitory control points present on their surface, such as the PD-1 receptor. Activation of these receptors takes place after combining with appropriate protein ligands (PD-L1), located on tumor cells. Preventing the interaction between PD-1 receptor and their ligands unblocks the activity of T lymphocytes and leads to the destruction of tumor cells. Immunotherapy with use of monoclonal antibodies blocking PD-1 receptors is starting to play a key role as one of the most effective and innovative cancer treatments in the last decade. The aim of the study was to determine the expression of PD-L1 in selected types of B-cell lymphomas. The study was retrospective, tissue material from 25 patients, 8 women and 15 men were qualified for the study. An immunohistochemical technique was used with the PD-L1 monoclonal antibody and archival tissue material embedded in paraffin from patients with diagnosed B-cell non-Hodgkin's lymphomas. It has been demonstrated that the expression of PD-L1 varies depending on the type of lymphoma. Diffuse large B-cell lymphoma shows a strong expression of PD-L1, and follicular lymphoma weak expression of PD-L1. The results suggest that PD-L1 antibody expression may be dependent on the tumor's malignancy and correlate with its clinical course.

162 P 109. The influence of Fe3+ on activity of cytostatic drugs used in colon adenocarcinoma treatment W. Stańco1, J. Kulbacka2

1Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, Wrocław, Poland 2Department of Molecular and Cellular Biology, Wroclaw Medical University, Wrocław, Poland [email protected] and [email protected] Purpose: Iron is a crucial microelement. Iron can exist in two forms: oxidized and reduced, what contributes to the formation of free radicals and chained reaction of lipid peroxidation [1]. The aim of the work was to assess how iron ions contained in Ferric citrate and Ethylenediaminetetraacetic acid iron(III) sodium salt affect the action of cytostatic drugs on human colon adenocarcinoma cells. Materials and methods: The studies were carried out on the human colon adenocarcinoma cells (LoVo). Cells were exposed to various concentrations of 5-fluorouracil (5-FU) for 24h and 48h. Then cells were exposed to 5-fluorouracil in combination with Ferric citrate and Ethylenediaminetetraacetic acid iron(III) sodium salt for 24h and 48h. Cell survival was determined by an MTT test after 24 and 48 hours incubation. Results: Our results show that adenocarcinoma cells activity decreased when we used 5-fluorouracil with iron ions, in comparison to treatment with 5-FU only. Conclusions: The obtained results proved that iron ions can act as anti-cancer factor in combination with some of cytostatic drugs. Thus, we suppose that iron ions can interact in additive or synergistic way with cytostatic drugs. The application of substances as ferric citrate solution or EDTA iron (III) sodium salt as a source of iron ions can have an anticancer potential. ACKNOWLEDGEMENTS The research was financed partially by the Student Scientific Group “Biology of cancer cells” (Wroclaw Medical University) and the project of Polish National Science Centre from the project SONATA BIS 6 (2016/22/E/NZ5/00671; PI: J. Kulbacka). REFERENCES: 1. Manz DH, Blanchette NL, et al. “Iron and cancer: recent insights” Ann N Y Acad Sci.;1368(1):149-161, 2016.

163 P 110. Self-preserving cosmetics – is it possible?

A. Starzec, M. Brożyna, F. Kominek, I. Fecka

Department of Pharmacognosy and Herbal Medicines, Wroclaw Medical University, Wroclaw, Poland Assuring microbial purity of cosmetics and personal care products is a major problem for the cosmetic industry. It is essential that cosmetics, as well as other products with the formulas based on water or organic compounds, contain some ingredients that prevents spoilage. Microbiological contaminations induce, among others, decomposition of active compounds or changes in physiochemical properties of cosmetics which may be harmful to customers' health. Thus, in order to prevent the growth of microorganisms, synthetic preservatives such as parabens, thiomersal, formaldehyde derivatives and Kathon CG are most commonly used. However, it is increasingly being discussed that those synthetic preservatives cause different allergic reactions and irritation of skin. Due to these reports, natural cosmetics has recently gained popularity. In such products synthetic preservatives have been replaced by various ingredients obtained mostly from plants that possess antimicrobial activity but are not legislated as preservatives according to Annex VI of Commission Directive 76/768/EEC. The aim of this work is to present the latest research works on natural substances (essential oils and plant extracts) that can be used as preservatives in self-preserving cosmetics production.

164 P 111. The anti-diabetic and antioxidant effects of purple potatoes (Solanum tuberosum L.) extract in streptozotocin-induced diabetic rats P. Strugała1, O. Dzydzan2, I. Brodyak2, I. Bila2, M. Liuta2, N. Sybirna2, J. Gabrielska1

1 Department of Physics and Biophysics, Wrocław University of Environmental and Life Sciences, Wrocław, Poland 2 Department of Biochemistry, Ivan Franko National University of Lviv, Lviv, Ukraine. Rich sources of substances having beneficial effect on human health are, among others, fruits and vegetables. One of the widely consumed vegetables in the world is the potato. Purple potatoes (PP) are a rich source of anthocyanins with multiple health effects. It has been already proved, that these relatively unknown and undervalued in Europe varieties contain high levels of anthocyanins in acylated forms. At variance to anthocyanins from fruits that are non-acylated, the acylated ones exhibit e.g. high thermal and hydrolysis resistance, which makes them technologically attractive, on top of health-beneficial properties. The aim of study was to evaluate effects of the PP extract, from variety Blue Congo, on blood glucose parameters and plasma oxidation stress markers in diabetic rats. Streptozotocin-treated Wistar male rats were given PP extract in dose 245 mg kg−1 bw. by intragastric administration for 2 weeks. The evaluation of oxidative stress in blood plasma was performed by measuring the content of products of lipid peroxidation, oxidative modificated proteins (OMPs), advanced – oxidation protein products (AOPPs) and glycosylation end-products (AGEs). The study has shown that the PP extract has significant antioxidant effect, decreasing MDA, OMPs, AOPPs and AGEs contents in blood plasma. Additionally, PP lowered both blood glucose and glycated haemoglobin, also improved glucose tolerance. In conclusion our study proved that the PP extract which has a high antioxidant potential, could be used as a promising drug or a dietary supplement for attenuation of such diseases as diabetes. ACKNOWLEDGEMENTS The research was financed by the (Polish) National Science Centre, Grant No.2017/25/N/NZ9/02915.

165 P 112. Content of heavy metals in beers from different countries D. Styburski1, M. Skórka-Majewicz1, W. Żwierełło1, J. Palma1, N. Komorniak1, J. Kikut1, J. Kałduńska1, A. Wolska1, P. Kupnicka2, E. Metryka2, P. Kapczuk2

1Department of Biochemistry and Human Nutrition, Pomeranian Medical University in Szczecin, Broniewskiego 24 street, 71-460 Szczecin, Poland 2Department of Biochemistry, Pomeranian Medical University in Szczecin, Powstańców Wlkp. av. 72, 70-111 Szczecin, Poland Introduction. Beer is a popular drink in many countries including Poland. Consumers often select national products, but imported beers are becoming increasingly popular. Content of heavy metals in beers is conditioned by concentration of elements in water used for production, which often has a different composition depending on the region and type of local yeast, malts grain and hops. Heavy metals cause changes in protein synthesis, ATP production and integrate with proteins, increasing accumulation of elements in body and inhibiting their elimination causing a many negative changes in body and development of diseases. Assumptions and purpose. The aim of the study is to provide an answer to question whether beer is a significant source of heavy metals for humans. Material and methods. 69 bottled beers from Poland and imported to Poland from Asia, South America, and European countries were used for the purpose of this study. The drinks were purchased in 2017. After degassing, beer samples were collected into plastic 20 ml tubes. The analysis of the liquid samples was investigated by ICP-AES. The statistical analysis was performed by means of the Statistica. The results were verified at the level of statistical significance p-value ≤ 0.05. Results. The study showed significant differences in the concentration of elements between beers from different countries. Conclusions. Beer is a significant source of heavy metals for the body. Chronic consumption of beer can lead to toxicity and in consequently have a negative effect on human health. REFERENCES: 1. Walker C.H., Hopkin S.P., Sibly R.M., Peakall D.B. (2002) “Fundamentals of ecotoxicology”, WN PWN, Warsaw 2002. 2. Preedy, V. R. (2009). “Beer in health and disease prevention”. London, UK: Academic Press. 3. Jarosz M. (2010) “A practical textbook on dietetics” Food and Nutrition Institute, Warsaw, Poland.

166 P 113. Novel eradication methods of bacterial biofilms in chronic diabetic foot infection Z. Sycz1, M. Sępek2, J. Jeżewska3, D. Wojnicz1

1Department of Biology and Medical Parasitology, Wrocław Medical University, Mikulicza-Radeckiego 9, 50-367 Wrocław, Poland 2Department of General, Minimally Invasive and Endocrine Surgery, Wrocław Medical University, Borowska 213, 50-556 Wrocław, Poland 3Department of Public Health, Division of Medical Social Sciences, Wrocław Medical University, Bartla 5, 51-618 Wrocław, Poland This paper focuses on the overview of novel methods applied for eradication of bacterial biofilms in chronic diabetic foot infection (DFI). The fundamental principles of DFI care are maximizing perfusion and off-loading strategies. The gold standard for biofilm eradication is Biofilm Based Wound Care (BBWC) – strategy designed to resolve the problem of bacterial biofilms in chronic non-healing DFI, such as wounds and ulcers. The main components of BBWC are appropriate wound bed preparation (mechanical debridement through removal of necrotic or devitalized tissue, wound exudates and even granulation tissue) and accompanying advanced treatment modalities based on anti-biofilm strategies. Anti-biofilm therapies are used in local wound treatment including: modern dressings (occlusive, semi-occlusive, absorbent, antimicrobial); silver (AgNO3, Ag7NO11 or sulfadiazine); chemical disinfectants (octenidine dihydrochloride, chlorhexidine digluconate also conjugated with gold nanoparticles, polyhexamethylene biguanide, povidone iodine, cadexomer iodine, hypochlorous acid); synthetic antibiotics (4th/5th generation cephalosporins, carbapenems, 3rd/4th generation fluoroquinolones, linezolid, daptomycin, triple antibiotic i.e. polysporin, nano-penicillin G, combination of gentamicin and L-arginine, calcium sulfate beads loaded with tobramycin or gentamicin, ultrasound and microbubbles gas-filled microstructures combined with gentamicin or streptomycin). Other methods used to treat biofilm infections in chronic wounds include: low intensity electric or electromagnetic field, radiofrequency electrical currents, pulsed electric fields, negative pressure wound therapy and combined with timed cyclical saline instillation, antimicrobial photodynamic therapy, nanoparticle-mediated photodynamic therapy, photodynamic antimicrobial chemotherapy, cold atmospheric plasmas, near-infrared laser, hyperbaric oxygen therapy, ultrasounds and freshly ozonated water. The novel approaches and therapies are currently under intensive investigation for their potential as antibiofilm agents, include: growth factors, granulocyte-stimulating factors, probiotics, dispersal signals (cis-2-decanoic acid), gallium, polyhydroxybutyrate, norspermidine, combination of caprylic acid with glyceryl trinitrate or with rhamnolipids, peptide-based (pexiganan), lactoferrin/xylitol hydrogel alone or in combination with the silver wound dressing, biosurfactants, natural origin substances (mentha, oregano, thyme, clove, tea tree, melaleuca essential oils and their constituents), medicinal honey, matrix targeting enzymes (N-acetyl-D- glucosamine-1-phosphate acetyl transferase, proteinase K, trypsin, dispersin B, KSL-W peptide, DNase, lactonase SsoPox-W263I), bacteriocins (nisin A, lacticin Q, nukacin ISK-1, NP101, NP108), antimicrobial peptides (cathelicidin/hCAP18, human β-defensin 3, dermcidin) and bacteriophage therapy.

167 P 114. Synthesis and potenial properties of N-substituted 2-hydroxy-3-(4-aryl-1- piperazinyl)propyl derivatives of Phthalimide D. Szkatuła1, P. Niziołek2

1Department of Chemistry of Drugs, Wrocław Medical University 2Student Scientific Club of Chemistry of Drugs, Wrocław Medical University Planty of research has proved that Phthalimide analogs devoid of the glutarimide ring, which contribute to the teratogenic activity, shown a wide range of pharmaceutical/biological activities [1, 2]. That’s why Phthalimide analogs are used as a superior seed compounds for creative new biologically active molecules [2, 3]. This study aimed to investigate on the the synthesis of N-substituted 2-hydroxy-3-(4-aryl-1-piperazinyl)propyl derivatives of Phthalimide, characteristic for previously described model structers [4]. It is expected that new derivatives may be considered as anti-inflammatory, analgesic and immunomodulatory agents. The structure of the obtained new Phthalimide derivatives was confirmed using IR Spectrometry, 1H NMR , 13C NMR and MS Methods.

168 P 115. Studies of spectroscopy techniques of 4-alkoxy-6-methyl-1H-pyrrolo [3,4-c]pyridine -1,3(2H)-diones on interaction with albumin E. Krzyżak1, D. Szkatuła2

1 Department of Inorganic Chemistry, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wrocław Medical University, 211A Borowska Str., 50-556 Wrocław, Poland 2 Department of Chemistry of Drugs, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wrocław Medical University, 211 Borowska Str., 50-556 Wrocław, Poland Using four techniques UV-VIS, spectroscopy, fluorescence spectroscopy, synchronous fluorescence spectroscopy and circular dichroism, the interaction of compounds with bovine serum albumin was examined. From the obtained results it can be concluded that the six tested imides are strongly bound to the albumin subdominal IIA, which causes the quenching in the intrinsic fluorensce of this protein by a static mechanism, thus suggesting the formation of stable ligand – albumin complex. This compounds slightly affects the change in the conformation of the secondary structure of the protein, by partially expending the chain and reducing the content of α-helix. As a result of binding, the polarity in the vicinity of the tyrosine residues in the albumin increases. The strength of influence of the compound with albumin was different for imides substituted or not substituted on the aryl ring (2-OCH3, 3-CF3). REFERENCES: 1. Guglielmelli A., Rizzuti B., Guzzi R. “Stereoselective and domain-specific effects of ibuprofen on the thermal stability of human serum albumin” European Journal of Pharmaceutical Sciences, 2018, 112, 122-131. 2. Peng W., Ding F., Xie Y.: “Biointeractions of C.I. Acid Red 2 and its structural analogues with transporter albumin: Fluorescence, circular dichroism, and ligand docking approaches” Journal of Photochemistry and Photobiology B: Biology, 2016, 154, 40-50. 3. Śladowska H., Filipek B., Szkatuła D., Sabiniarz A., Kardasz M., Potoczek J., Sieklucka-Dziuba M.., Rajtar G., Kleinrok Z., Lis T., et al. “Investigations on the synthesis and pharmacologicalproperties of 4-alkoxy-2-[2-hydroxy-3-(4-aryl-1-piperazinyl)propyl]-6-methyl-1H-pyrrolo[3,4-c]pyridine-1,3(2H)- diones” II Farmaco, 2002, 57, 897-908.

169 P 116. The potential of new vaccines in the treatment of fentanyl and oxycodone abuse B. Szydłowska, M. Szmidt, A. Wachowska, E. Walasek

Student Science Club of Department of Toxicology, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, Borowska 211, 50-556 Wrocław [email protected], [email protected], [email protected], [email protected] Opioid drugs are important tools to alleviate pain of different origins, but they have strong addictive potential. Fentanyl is an extremely potent synthetic opioid that has been increasingly used to adulterate heroin, cocaine, and counterfeit prescription pills, leading to an increase in opioid-induced fatal overdoses. Oxycodone, also syntetic opioid is full agonist of the μ-opioid receptor – oxycodone's effects are strikingly similar to heroin and narcotic effects are already in therapeutic doses. A vaccine targeting opioids (e.g. fentanyl or oxycodone) could offer protection against the toxic effects of fentanyl and oxycodone. Currently, innovative vaccines are being intensively investigated. The latest literature data on vaccines relevant to the treatment of addiction has been presented. Opioid vaccines contain modified form of the target opioid, chemically conjugated to a protein (OXY-KLH and F-KLH). It will stimulate immune system to produce antibodies that will be selective for the target opioid. When the opioids bind to the antibody, they are no longer active. This complex is too big to cross the blood-brain barrier, so it prevents its distribution to the central nervous system and its interaction with opioid receptors, which block addictive effect of opioids. Vaccines can also block toxic effects of opioids: respiratory depression, coma or bradycardia. Therapeutic vaccines have shown efficacy in a wide range of preclinical models and suggest that vaccines may offer a therapeutic option in reducing fentanyl and oxycodone-induced side effects, but the road from the laboratory to the clinic is still long. REFERENCES: 1. Hwang S.C.et al. “Efficacious Vaccine against Heroin Contaminated with Fentanyl” ACS Chem Neurosci. 2018 June 20; 9(6): 1269-1275. 2. Pravetoni M, Vervacke JS, Distefano MD, Tucker AM, Laudenbach M, Pentel PR (2014) “Effect of Currently Approved Carriers and Adjuvants on the Pre-Clinical Efficacy of a Conjugate Vaccine against Oxycodone in Mice and Rats” PLoS ONE 9(5): e96547. 3. Raleigh MD, Peterson SJ, Laudenbach M, et al. “Safety and efficacy of an oxycodone vaccine: Addressing some of the unique considerations posed by opioid abuse” PLoS One. 2017;12(12):e0184876. Published 2017 Dec 1. doi:10.1371/journal.pone.0184876. 4. Olson ME, Janda KD. “Vaccines to combat the opioid crisis: Vaccines that prevent opioids and other substances of abuse from entering the brain could effectively treat addiction and abuse” EMBO Rep. 2017;19(1):5-9.

170 P 117. Innovative method of treating Parkinson's disease. The implant that gives patients new life K. Szwed1, K. Synowiec1, A. Tomczak1, E. Sawicka2

1Student Science Club of Department of Toxicology, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, Wroclaw, Poland 2Department of Toxicology, Faculty of Pharmacy, Wroclaw Medical University, Wroclaw, Poland Parkinson's disease is a degenerative disease of the central nervous system. Symptoms accumulate over the years. Initially, patients have slight mobility and writing problems, in the advanced phase of the disease, the simplest activities, such as eating or dressing, are impossible for them. Specialist from California University have created the newest neurostimulation device that enables brain activity stimulation and registering it in real-time. The “WAND” – wireless artifact-free neuromodulation device - consists of two small-sized external controllers and both of them monitor sixty four electrodes located in the brain. This wireless device monitors electrical activity in the brain identifying wrong signals, which indicate the appearance of convulsive seizure or shaking. This technology uses nonstandard ASIC system both for neural and electrical registration. WAND models electrical signals in the brain in response to patient’s neurological condition, preventing negative events and symptoms of neurological diseases. The systems that have existed until now are limited by the low number of channels, the lack of algorithmic flexibility and the distortion of registered signals. In contrast, WAND is able to register electrical activity from 128 different channels, and it also has a unique construction registering even the slightest signals emitted by the brain. Due to insufficient effectiveness of the currently used methods, new methods of treatment are constantly being sought after. WAND provides an opportunity for progress and a new direction in the treatment of Parkinson's disease, epilepsy and other neurological disorders.

171 P 118. Laboratory evaluation of CLL A. Ślęzak1, A. Wysoczańska-Klaczyńska2, M. Adamska3, K. Dmochowska3, A. Konias3, M. Terpińska3, R. Ryczan-Krawczyk1, K. Gajek1, M. Ussowicz1

1Department and Clinic of Paediatric Bone Marrow Transplantation, Oncology and Hematology, Wroclaw Medical University, Wroclaw, Poland 2Department of Basic Medical Sciences, Wroclaw Medical University, Wroclaw, Poland 3Department of Laboratory Diagnostics, University Teaching Hospital, Wroclaw, Poland Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in Western populations and is characterized by monoclonal B cell proliferation. Incidence in males is higher than in women with a M:F ratio of 1.7:1, and ethnic-related genetic predisposition is found in Caucasians. The median age at diagnosis is 70 years but the disease can be found in individuals at younger age. Pathognomic for CLL are smudge of lymphocytes in peripheral blood smear. The flow cytometric immunophenotyping of circulating lymphocytes in CLL shows the abnormal coexpression of B-cell antigens such as CD19, CD20, CD23, and T-cell antigen CD5. In addition, surface membrane immunoglobulins are detected at low level, usually of IgM or both IgM and IgD class. The initial symptoms of CLL can be faint. Some patients may notice non-tender swelling of lymph nodes. The most common physical examination finding is lymphadenopathy, less frequently splenomegaly and hepatomegaly.The accidental finding of lymphocytosis could initiate the diagnostic process. Anemia and thrombocytopenia at diagnosis can occur in some patients defining more advanced stages of the disease. In advanced stage, the number of lymphocytes may exceed one hundred thousand and be difficult to reduce or control. Bone marrow aspiration and biopsy are not necessary to make a diagnosis. The treatment is recommended in symptomatic patients or in rapidly progressing disease. The aim of the study is to present laboratory parameters occurring in CLL based on Wroclaw Teaching Hospital Labolatory experience. Also assessment of the characteristic microscopic smears, staging of the symptoms and most common features of CLL are shown. REFERENCES: 1. G. Ferrer, et al. Molecular Medicine vol. 1 p. 24-9, 2018. 2. O. Al-Sawaf, et al. Oncology Research and Treatment vol. 39 p. 768-778, 2016. 3. P. Bose, et al. F1000Research vol. 6 p. 1924, 2017. 4. M. Hallek, Annual Clinical Updates in Hematological Malignancies vol. 92 p. 946-965, 2017.

172 P 119. The effect of cyclophosphamide on the binding of imatinib to human holo-transferrin. The spectroscopic studies U. Śliwińska-Hill

Department of Analytical Chemistry, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wrocław Medical University, Wrocław, Poland [email protected] The interactions of proteins with drugs are very important from the pharmacological point of view. Human holo-transferrin (hTf) main function is iron-binding and the metal transport. Moreover, the protein is able to bind and transport various endogenous and exogenous ligands, especially drugs. Imatinib mesylate (Imt) is a tyrosine kinase inhibitor mainly used in the treatment of blood cancers, frequently in the multidrug therapy with cyclophosphamide (CYP). In this study the effect of cyclophosphamide on the interaction of imatinib mesylate with human holo-transferrin has been investigated. Using spectroscopic techniques such as fluorescence, circular dichroism (CD), ultraviolet-visible (UV-VIS) and electrophoretic light scattering (ELS) the binding affinity, thermodynamic parameters, system stability and effect of ligands on the holo-transferrin secondary structure at varying pH values has been determined. Calculated binding constants are on the order of 104-106 M-1 but they are higher under physiological conditions (pH 7.40). Determined thermodynamic parameters show that hydrophobic and ionic strength are predominant under physiological and alkaline conditions and van der Waals and hydrogen bonds are involved in the interaction in the acid environment. The CD studies show that there are some changes in the α-helical structure of protein after interaction with imatinib and cyclophosphamide. Zeta potential measurements proved that all systems are unstable under tested conditions. This study should be helpful for realizing the distribution and transportation of the drugs in the combination therapy as well be of great use for understanding the effectivity of the multi drug therapies.

173 P 120. The use of modern technologies by Polish dentists M. Świtała

Wrocław Medical University, Student Group of Experimental Dentistry and Biomaterials Research, Wrocław, Poland The aim of this study was to investigate the use of modern technologies by Polish dentists in different dental procedures, future use, dentist’s opinion about effectiveness of mentioned techniques and correlation between use of modern technologies and factors like work experience, sex, size of office. As ,,modern technologies” considered were: dental laser, ultrasounds, air abrasion, ozonoterapy, caries diagnostics methods, chemo-mechanical caries removal, pulp vitality tests and computer-controlled local anesthetic delivery (CCLAD). The questionnaire was posted in social-media group of Polish dentists. 112 dentists were included in the study. 99,11% questioned dentists use at least one of modern technologies. The most use ultrasounds (97,32%). 84,82% of respondents use technologies other than ultrasounds. 42,86% questioned dentists use CCLAD. Dental laser was used by 33,04% of dentists, innovative caries diagnostics methods by 32,14%, air abrasion by 28,57%, chemo-mechanical caries removal by 21,4%, innovative tests for pulp vitality by 17,8%. There was correlation only between use of dental laser and size of office (p=0,015) and between use of CCLAD and size of office (p=0,048). The most common cause for not using mentioned technologies was their high cost (50,89%). 98,21% of respondents claim that they will use modern technologies (excluded ultrasounds) in the future. Many Polish dentists tends to use innovative technologies in dental practice. The most common of them are ultrasounds. Other techniques aren’t popular among Polish dentist because of their high cost. Generally, use of modern technologies doesn’t depend on work experience, sex and size of office.

174 P 121. Impact of polyethylenimine on cell viability in culture of normal and cancer cells K. Tądel1, K. Wakulik1, B. Wiatrak2, H. Moreira2

1 Student Research Group of Flow Cytometry and Biomedical Research, Wroclaw Medical University, Wroclaw, Poland 2Department of Basic Medical Science, Wroclaw Medical University, Wroclaw, Poland [email protected] Polyethylenimine (PEI) is a synthetic cationic polymer containing repeating units of amino groups. PEI can exist as linear or branched polymer depending on form of amino groups present in its structure. If all of the amines are secondary, then the polymer is linear. The PEI polymer has a branched structure when primary, secondary and tertiary amines are present. Three new polyethylenimines with branched structure, denoted by PEI A, PEI B, PEI C, were synthesized at Wroclaw University of Technology. The aim of this study was to evaluate the cytotoxicity of these polymers in normal and cancer cells. Normal human dermal fibroblasts (NHDF cell line), drug-sensitive and drug-resistant colon cancer cells (LOVO and LOVO/DX cell lines, respectively) were used in the study. Cell culture plates were coated with newly synthesized polymers at concentrations of 50, 100 and 500μg/ml for 30 min at RT. Then, the cells were seeded into coated plates and incubated for 24, 48 or 72 hours at 37°C and 5% CO2. The cytotoxicity was evaluated using MTT assay. PEI A and PEI B did not affect NHDF cell viability in the whole range of concentrations tested while PEI C exhibited cytotoxic activity at 50 and 100μg/ml. All polymers caused statistically significant decrease in viability of both LOVO and LOVO/DX cells. This effect was proportional to the concentrations tested. The cytotoxic effects were not significantly dependent on the time of incubation. All newly synthesized PEI polymers show cytotoxicity on cancer cells, but have no cytotoxic activity on normal cells excepting PEI C. The observed different effects of PEI polymers on normal and cancer cells probably result from differences in their structure.

175 P 122. First application of nano-electrochemotherapy in feline oral malignant melanoma treatment J. Tunikowska1, J. Kulbacka2, A. Antończyk1, V. Novickij3, N. Rembiałkowska2

1Department of Surgery, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, Wroclaw, Poland 2Department of Molecular and Cellular Biology, Wroclaw Medical University, Wroclaw, Poland 3Institute of High Magnetic Fields, Vilnius Gediminas Technical University, Vilnius, Lithuania [email protected], [email protected] Melanoma of the oral cavity is rare in cats, and its diagnosis is usually associated with poor prognosis [1]. Local control of this tumor requires wide surgical excision with additional medical treatment [2]. A 16-year-old European cat was diagnosed with malignant oral melanoma. The tumor was approximately 2 cm in diameter and was located at the level of the premolar teeth of the maxillary bone involving cheek wall and lip angle. Tumor was prone to multiple injuries and bleeding. Difficulties in food intake and cachexia was observed. Due to the senile age of the patient, as well as chronic renal failure, radical surgery or radiotherapy was declined by the owner. Surgery was conducted under a short infusion anesthesia and local anesthesia of the maxillary nerve. The tumor was marginally removed with a CO2 laser (Eraser C) working in continuous mode and 10W of power output. The excisional site was injected with bleomycin and nano- electroporated. For pulse delivery, the PPG-20 generator (FID Technology, Germany) and an applicator with needle-type (9 mm gap) steel electrodes were used. A 15±4 kV/cm x 15 ns pulsing protocol was employed and a total of 800±100 pulses were delivered to the site at repetition frequency of 200 Hz. Intraoperatively, no negative tissue effects were observed as well as no involuntary skeletal muscle contractions that are characteristic for electrochemotherapy. The patient received buprenorphine at a dose of 20 mcg / kg every 8 hours for the first 3 days. Amoxicillin with clavulanic acid at a dose of 20 mg / kg was administered every 12 hours for two weeks. Single injection of a dexamethasone at a dose of 1mg / kg was given to reduce massive swelling of the upper lip and cheek that occurred at the second day after surgery. Three days after the surgery, appetite restoration and normal food intake was noticed. During the next two weeks areas of necrosis together with areas of normal heeling were observed. Despite the promising treatment effects during the perioperative period, further observation of the long-term effects of nano-electrotherapy is necessary. ACKNOWLEDGMENTS The study was realized with the financial support of the project of Polish National Science Centre from the project SONATA BIS 6 (2016/22/E/NZ5/00671; PI: J. Kulbacka). REFERENCES: 1. Stebbins KE, Morse CC and Goldschmidt MH. “Feline oral neoplasia: a ten-year survey” Vet Pathol Online 1989; 26: 121-128. 2. Chamel G., Abadie J., Albaric O., Labrut S., Ponce F., & Ibisch C. “Non-ocular melanomas in cats: a retrospective study of 30 cases” JFMS 2017; 19(4): 351-357.

176 P 123. Cytotoxic effect of Celastrol and Camptothecin on drug-resistant and drug-sensitive cancer cells K.Wakulik1, K.Tądel1, M. Pawelska1, K. Młynarska1 , J. Piasny1 , B. Wiatrak2, H. Moreira2

1 Student Research Group of Flow Cytometry and Biomedical Research, Wroclaw Medical University, Wroclaw, Poland 2Department of Basic Medical Science, Wroclaw Medical University, Wroclaw, Poland [email protected] Celastrol is a plant triterpene isolated from the root of Chinese herb Tripterygium wilfordii. This polyphenol has various beneficial therapeutic properties, including anticancer activity. Camptothecin is a quinolone alkaloid of extracts from the Chinese tree Camptotheca acuminate which shows anticancer activity mainly by inhibiting the DNA I topoisomerase. The aim of this study was to investigate the cytotoxic effects of both Celastrol and Captothecin on drug resistant cancer cells and cancer cells displaying standard sensitivity to cytotoxic treatment. The study was carried out with doxorubicin resistant human carcinoma cell lines: LOVO/DX (colon cancer cells) and MCF-7/DX (breast cancer cells) and drug sensitive cell lines: LOVO, MCF-7 and A549 (lung cancers). Celastrol and Camptothecin were used at various concentrations ranging from 0,78µM to 100µM. The cells were incubated for 24 hours in the presence of the tested drugs. The cell viability was assessed by the means of MTT assay. Our study demonstrated that Celastrol and Camptothecin affect cell viability in all cancer cells used in the study. The cytotoxic effects was dose depended and comparable for both compounds. Although, Celastrol showed significantly stronger cytotoxic effects against MCF7/DX cells compared to Camptothecin. In addition, there were no significant differences in their effect on drug-resistant and drug-sensitive cells. Celastrol and Camptothecin demonstrate a strong cytotoxic effects on colon, breast and lung cancer cells. This effect is comparable in the drug-resistant and drug-sensitive tumor cells, which suggests their effectiveness in the treatment of cancers with various disease severity. In further studies, the possible synergistic effect of both compounds will be evaluated.

177 P 124. Alternative aproaches of curring spinal muscle atrophy M. Wastag2, W. Szlasa1, M. Ważna2, M. Knura1, M. Gebuza1

1 Faculty of Medicine, Wroclaw Medical University, Poland 2 Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, Poland Spinal muscle atrophy (SMA) is a common disease, characterized by loss of lower motor neurons and progressive muscle wasting. The problem contributes also to the neuromuscular junctions which are highly affected by the accumulation of neurofibrillary tangles in presynaptic neurons. The disorder is based on a defects of SMN genes, which codes a chaperon protein. The chaperon is crucial in forming a spliceosome complex with Germin 2 and 8. The lack or defect of SMN gene causes the disturbances on the posttranscriptional level, that leads to neuromuscular disorders. The only registered by FDA drug for SMA treatment costs 80 000$ for an ampule. The aim of this project was to gather information about alternative drugs, which could be useful in SMA treatment[1]. There have been analyzed valproic acid (VPA), Salbutamol (Albuterol) in order to find weather these could be useful in therapy. Research made in 2005-2007 suggest, that VPA is not as effective as it was thought to be. Nowadays the scientists are focused on Salbutamol, which strengthens the muscle tension and dilates bronchi. First results gives us high hopes of finding a low price, highly effective medicine for SMA treatment. The project not only focuses on the genetics and molecular basis of the disorder, but also on pharmacoeconomics of the problem. ACKNOWLEDGMENTS The work was created as part of the activity of the Student Research Group “Biology of Cancer Cell” at the Wroclaw Medical University (SKN No. K 148). REFERENCES: 1. http://www.curesma.org/research/our-strategy/drug-discovery/therapeutic-approaches/

178 P 125. Spectroscopic and molecular docking studies on the interaction between vitamin B9 and human hemoglobin K. Wiglusz1, R.J. Wiglusz2

1Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, Borowska 211 A, 50-566 Wroclaw, Poland 2Institute of Low Temperature and Structure Research, Polish Academy of Sciences, Okolna 2, 50-422 Wroclaw, Poland Vitamin B9, also known as folic acid (pteroyl-L-glutamic acid, folate, folacin), is an organic compound belonging to a group of folates showing biological activity. Its deficiency could be the cause of disorders in many important metabolic processes, e.g. in the synthesis of nucleic acids, proteins, amino acids, neural tube defects. In the present work, there have been studied the interactions between vitamin B9 and human hemoglobin by different spectroscopic methods (fluorescence technique, circular dichroism (CD), dynamic light scattering (DLS) and FTIR spectroscopy) as well as molecular modelling method. It has been found that vitamin B9 quenched the fluorescence of protein due to the formation of the complex as a static quenching mechanism with binding constants of 1.53 (±0.01) ∙ 105 and 0.73 (±0.03) ∙ 104 M-1 at 300 and 310 K, respectively. Noncovalent bond types e.g. electrostatic interactions, hydrogen bonds, van der Waals and hydrophilic interactions play major roles in the binding of folic acid to hemoglobin. It has been noted that the folic acid caused microenvironmental perturbations around the tyrosine residues and slightly effected on secondary structure changes in protein at physiological conditions pH 7.4. The isoelectric point of hemoglobin-folic acid complex has been indicated at pH 6.64 and its higher than the pure solution of protein. Taking into account the above-mentioned aspects the folic acid can be bound by the human hemoglobin affecting its structural and functional properties, and providing a new insights into the binding mechanism.

179 P 126. Bromolactones as potential candidates in the chemotherapy of cancer: their antiproliferative activity and the effect on the properties of biological membranes A. Włoch1, W. Gładkowski2, M. Mazur2 A. Pawlak3, B. Obmińska-Mrukowicz3, H. Kleszczyńska1

1Department of Physics and Biophysics, Wrocław University of Environmental and Life Sciences, Wrocław, Poland 2Department of Chemistry, Wrocław University of Environmental and Life Sciences, Wrocław, Poland 3 Department of Pharmacology and Toxicology, Wrocław University of Environmental and Life Sciences, Wrocław, Poland Searching for the active compounds as potential drug candidates in the chemotherapy of cancer chiral bromolactones possesing a 2,5-dimethylphenyl substituent has been synthesized. The subject of the study were both enantiomers of cis δ-bromo-γ-lactones and trans δ-bromo-γ- lactones. The studies on interactions between anticancer drugs and cell membranes is an important area and can help to explain mechanism of drug action. Therefore, the aim of the research was to determine the antiproliferative and hemolytic activity of bromolactones and their effect on the properties of biological membranes. Antiproliferative activity was determined on three canine cancer lines: D-17 (canine osteosarcoma), GL-1 (B cell leukemia) and CLBL-1 (B cell lymphoma) and one human (Jurkat) using the MTT test. The hemolytic activity test was performed on red blood cells (RBCs) and was based on the concentration of hemoglobin released from erythrocytes. The effect of bromolactones on the properties of biological membranes was determined using fluorescent probes like MC540, Laurdan, and DPH located in different areas of the membrane. Our results indicate that tested compounds do not have a toxic effect on red blood cells but they showed significant cytotoxicity against canine and human cancer cell lines. These compounds concentrate mainly in the hydrophilic part of erythrocyte membrane but have practically no influence on fluidity in the hydrophobic region. Our preliminary studies indicate that tested cis and tans isomers of δ-bromo-γ-lactones are potentially good candidates as anticancer drugs and could be used in future in pharmacological preparations. ACKNOWLEDGEMENTS: Work supported by Wroclaw Centre of Biotechnology, programme The Leading National Research Centre (KNOW) for years 2014-2018 and funds of statutory activities of the Department of Physics and Biophysics, Wroclaw University of Environmental and Life Sciences. REFERENCES: 1. W. Gładkowski, et al. Molecules, 23, 3035, 2018.

180 P 127. Improving bowel function in two weeks thanks to a small dietary intervention - the results of two weeks project A. Wolska1, H. Mruk1,2, N. Komorniak1, J. Kikut1, W. Żwierełło1, M. Majewska-Skórka1, J. Palma1, D. Stryburski1, A. Gudan1, O. Fryda3, D. Matyniak3, M. Popik3, M. Kaczmarek1, N. Bajur1, D. Maciejewska1, K. Skonieczna-Żydecka1, E. Stachowska1

1 Department of Biochemistry and Human Nutrition, Pomeranian Medical University in Szczecin, Poland 2 Faculty of Medicine, Pomeranian Medical University in Szczecin, Poland 3 Faculty of Biotechnology, Pomeranian Medical University in Szczecin, Poland Prebiotics are indigestible nutrients that are a source of energy for intestinal microbiota. During the bacterial fermentation of prebiotic substances short-chain fatty acids (SCFA) are formed whose function is to maintain homeostasis in the human body, as well as affect the functioning of the immune system. The concentration of short-chain fatty acids may be important in inflammatory diseases, allergies or intestinal dysbiosis caused by antibiotic therapy. To assess the impact of a 14 day prebiotic bar, containing 6 or 12 g fiber (different fractions) on the frequency of bowel movements and and on SCFA content in feces. Analyzing the content of SCFAs, it was noticed that between the groups receiving both 6g fiber and 12g fiber, there was an increase in the content of all SCFAs tested regardless of the content of fiber in bars. There was also a strong trend of SCFA content increase in the group of women using a bar with a fiber content of 12 g, but it was not statistically significant. In the group of people consuming a bar with a content of 12 g, the average SCFA content was 234.94 mmol / kg and in the group of women consuming control bars 125.91 mmol / kg. Analyzing the 10-point scale VAS intensity of gastrointestinal symptoms of study participants before and after the intervention, no significant changes in the intensity of abdominal pain were noted (Before: 0.5 PO: 0.583; p = 0.75), flatulence (Before: 1.556 PO: 0.833; p = 0.44), gases (Before: 1.778 PO: 1.083, p = 0.41), nausea (Before: 0.0 PO: 0.556, p = 0.11), bounce (Before: 0.5 PO: 0.056, p = 0.01) and feelings of incomplete bowel movement (Before: 0.33 PO: 0.33; p> 0.99). The mean number of defecations before the intervention was 0.28 (2/7 days), and after 2 weeks of the test 0.872. This difference was statistically significant (p = 0.0002). The current intake of macronutrients did not differ significantly after the use of intervention compared to the beginning of the study. However, significantly lower intake of protein and higher fiber was observed after 2 weeks of intervention. Suplementacion of prebiotics induces significant changes in the concentration of short-chain fatty acids in feces (in women consuming 12g fibers they were twice as high). Increased fiber intake increases the number of bowel movements, without additional side effects such as abdominal pain, bloating or constipation.

181 P 128. The anthropometric parameters in survivors of acute lymphoblastic leukemia A. Wysoczańska-Klaczyńska1, A. Ślęzak 2, B. Wiatrak1, S. Płaczkowska3, E. Barg1

1Department of Basic Medical Sciences, Wroclaw Medical University, Wroclaw, Poland 2Department and Clinic of Paediatric Bone Marrow Transplantation, Oncology and Hematology, Wroclaw Medical University, Wroclaw, Poland 3Diagnostics Laboratory for Teaching and Research, Wroclaw Medical University, Wroclaw, Poland Acute lymphoblastic leukemia (ALL) is the most common type of childhood cancer. One of the negative consequences of ALL treatment may be the occurrence of late effects, including growth and weight disorders. The aim of the study was to evaluate the anthropometric parameters of patients treated for ALL during childhood and adolescence. The study group consisted of 50 survivors aged 5-18 (26 boys), at least 1 year after successful ALL treatment. Control group consisted of 60 participants aged 12.1±3.5 (30 boys). Weight, height, and BMI measures were converted into standard deviation scores (SDS). It was assumed that a short stature is for SD [<-1.88], overweight and over-height SD between [<+1.66- +1.88>], obesity and tall stature SD [>+1.88]. The study group and the control group were compared by using test-T. Weight disorders occurred in 15 survivors. The highest WSDS reached +5.97. In the control group 11 children were obese. Short stature was observed in 9 survivors and in 3 in the control group (15.20% vs. 5.45%). In the girls group, the more time after ALL therapy the more short stature symptoms were observed (0.12 vs -0.5). The survivor boys were significantly shorter compared to the control group (p=0.027). Boys survivors, but not girls, after successful ALL treatment were significantly heavier than the control group (p=0.046). In the examined group BMI SDS was higher than BMI SDS in control group but not significantly. For the group of boys at least five years after ALL therapy, a significant difference was observed in HSDS results compared to the control group (p=0.048).

182 P 129. Biotransformation of loxoprofen analogue A. Zagała1, M. Mazur1

1Department of Chemistry, Wrocław University of Environmental and Life Sciences, Norwida 31, 50-375 Wrocław, Poland The α, β-unsaturated ketones exhibited wide range of biological activities like antifungal, antibacterial, antiviral, anticancer, anti-inflammatory and antioxidant [1-3]. Microbial transformations of those compounds can serve as the model processes to investigate their metabolism in living organisms. The biotransformations are also the useful tool to obtain biologically active derivatives with additional functional groups or new stereogenic centers. These structural modifications can be crucial for increasing biological activity. The substrate for biotransformation was α, β-unsaturated ketone (1) with p-metoxyphenyl substituent, obtained in aldol condensation reaction between cyclopentanone and p- anisaldehyde. The biotransformation processes were carried out using whole-cell biocatalysts. Among ten tested microorganisms Candida parapsilosis AM909 and Fusarium avenaceum AM12 were able to effective substrate transformation. Two selected strains catalysed the reduction of double bond. In all transformation processes the corresponding ketone (2) was obtained as the only product. The structure of obtained compound (2) is similar to loxoprofen – a non-steroidal anti-inflammatory drug.

REFERENCES: 1. H. I. El-Subbagh, et al. J. Med. Chem. vol.43 p. 2915-292, 2000. 2. E. Erciyas, et al. J.Pharm. Sci. vol. 83 p. 545-548, 1994. 3. Y. Eryanti, et al. Makara, Sains vol.15 p. 117-123, 2011.

183 P 130. Current view on stem cells in regenerative dentistry W. Zakrzewski1, M. Dobrzyński1, M Szymonowicz1, K Kołodziejczyk1, Z. Rybak1 1Wroclaw Medical University, Department of Experimental Surgery and Biomaterials Research, Poland Nowadays, stem cell therapy has become a very promising scientific research topic. Although teeth are a very challenging material for regenerative medicine, they have the advantage to be the most natural and non-invasive source of stem cells. Thanks to their osteogenic and chondrogenic potential, they can be used in both oral and extra-oral regenerative therapy. Stem Cells of Human Exfoliated Deciduous Teeth can be even useful in treating neural deficits, which means, that developing dental regeneration therapy simultaneously significantly influences general medicine. Partially successful regeneration of tooth was also achieved with the use of extra-oral stem cells, which can lead to non-invasive and relatively cheap regenerative therapy in the future. Although stem cell use has still challenges to overcome, it is a very promising, cutting-edge therapy; its versatility can be a turning point for future dentistry and general medicine. REFERENCES: 1. Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc NatlAcadSci. december 2000, Vol. 97(25), pp. 13625-30, DOI: 10.1073/pnas.240309797. 2. Jain, Ramta Bansal and Aditya. Current overview on dental stem cells applications in regenerative dentistry. J Nat Sci Biol Med. January-June 2015, Vol. 6(1), pp. 29-34, [doi:10.4103/0976-9668.149074]. 3. Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. 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Comparative analysis of in vitro osteo/odontogenic differentiation potential of human dental pulp stem cells (DPSCs) and stem cells from the apical papilla (SCAP),. Arch Oral Biol. July 2011, Vol. 56(7), pp. 709-21, doi: 10.1016/j.archoralbio.2010.12.008. . 12. Friedlander LT, Cullinan MP, Love RM.IntEndod, J 2009 and 42:955-62. Dental stem cells and their potential role in apexogenesis and apexification. Int Endod J. 2009, Vol. 42, pp. 955-962. 13. Cai J, Zhang Y, Liu P, Chen S, Wu X, Sun Y, Li A, Huang K, Luo R, Wang L, Liu Y, Zhou T, Wei S, Pan G, Pei D, 2013 Jul 30 and 2(1):6. Generation of tooth-like structures from integration-free human urine induced pluripotent stem cells. Cell Regen (Lond). July 30, 2013, Vol. 2(1), pp. 6, doi: 10.1186/2045-9769-2-6. 14. Fa-Ming Chen, corresponding author# Li-Na Gao, Bei-Min Tian, Xi-Yu Zhang, Yong-Jie Zhang, Guang-Ying Dong, Hong Lu, et. al. 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184 P 131. Reorganizations in the level of metalloproteinases MMP-2 and MMP-9 and their inhibitors in rats' brain in the model of inflammation caused by fluoride W. Żwierełło1, M. Skórka-Majewicz1, D. Styburski1, J. Kałduńska1, J. Kikut1, A. Wolska1, N. Komorniak1, J. Palma1, P. Kupnicka2, A. Łukomska1,3

1 Department of Biochemistry and Human Nutrition, Pomeranian Medical University in Szczecin, Broniewskiego 24, Szczecin 71-460, Poland 2 Department of Biochemistry and Medical Chemistry, Pomeranian Medical University in Szczecin, Powst. Wlkp. 72, Szczecin 70-111, Poland 3 Laboratory of Neuroplasticity, Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur Street, 02-093 Warsaw, Poland Matrix metalloproteinases are versatile endopeptidases with many different functions in the body in health and disease. In the brain, matrix metalloproteinases are critical for tissue formation, neuronal network remodelling, and blood–brain barrier integrity. Fluoride is a potent neurotoxin that disrupts the function of neurons and causes a wide range of long-term side effects, including belong to disorders memory, concentration and learning, especially in young, developing individuals. The aim of the study is to investigate the influence of chronic inflammation on the level of MMP-2 and MMP-9 metalloproteinases and their specific inhibitors TIMP-2 and TIMP-3 in selected structures of the developing rat brain. The studies were carried out on 12 Wistar rats. Pregnant females, and later their offspring's was administered sodium fluoride in drinking water until reach sexual maturity. After the experiment, the animals (mature - 3 months) were killed, and their brains were used for further research. Fluoride already in low concentrations affects the synthesis of MMP-2 and MMP-9. Statistically significant increase in MMP-2 concentration was observed in the prefrontal cortex, striatum and cerebellum, as well as the decrease in MMP-9 concentration in the cerebellum with respect to control. The TIMP-2 level was lower in all subjects' structures in the study group, TIMP-3 was significantly lower (65%) in the cerebellum. Based on the results of the tests carried out, it can be concluded that fluoride affects for the synthesis of all analysed enzymes and their endogenous inhibitors, which can cause the formation of cognitive disorders.

185

186 INDEX OF AUTHORS

Adamczyk Z...... 34 Czyżnikowska Ż...... 94, 105, 149 Adamska M...... 172 Dąbrowski P...... 64 Alama A...... 55 Dec K...... 157 Antończyk A...... 176 Dębiński M...... 65 Azarov J...... 25 Dmochowska K...... 172 Backiel S...... 139 Dobryszycki P...... 30, 154 Bajur N...... 181 Dobrzyński M...... 51, 52, 184 Banaszkiewicz M...... 49, 133 Dolińska B...... 140, 151 Bania J...... 61, 131 Drąg M...... 21, 77, 95 Bański M...... 116 Drożdż A...... 36 Bar J...... 65 Drzozga K...... 66 Barański J...... 80 Dudek K...... 52 Barg E...... 50, 130, 182 Dumnicka P...... 56 Bartoszewicz M...... 68, 141 Durślewicz J...... 48 Bartuzi Z...... 48 Dyba A...... 67 Bednarska-Chabowska D...... 72 Dydak K...... 68, 141 Belik V...... 79 Dyrcz M...... 138 Benkowska D...... 29 Dzięgiel P...... 31 Berghausen-Mazur M...... 118 Dzydzan O...... 165 Biedroń A...... 56 Fecka I...... 58, 164 Bielak K...... 30 Franczak A...... 111, 112 Bila I...... 165 Fryda A...... 92 Bil-Lula I...... 49, 111, 112, 133 Fryda O...... 181 Bird P...... 95 Gabrielska J...... 165 Bis G...... 50 Gajek K...... 172 Bizoń A...... 158 Gebuza M...... 178 Błaszczyk M...... 57 Geneja M...... 69 Bodalska A...... 58, 101 Glatzel-Plucińska N...... 160 Bogacz-Radomska L.43, 59, 75, 76, 84, 85, 135, Gliszczyńska A...... 70, 132, 150 136, 138, 139 Gładkowski W...... 180 Bonarska-Kujawa D...... 125 Gładysz O...... 71 Bosiacki M...... 124, 155 Głogowska N...... 72 Boszkiewicz K...... 101 Gomułkiewicz A...... 31 Bovenschen N...... 77, 95 Grelich M...... 73 Brand I...... 45 Grzegrzółka J...... 31 Brasuń J...... 78, 86 Grzelak J...... 64 Brodyak I...... 165 Grzeszczak A...... 74 Brożyna M...... 114, 145, 164 Gubernator J...... 74 Bugla-Płoskońska G...... 61, 131 Gudan A...... 181 Bystranowska D...... 156 Gula D...... 94 Bystroń J...... 61, 131 Harasym J.43, 59, 75, 76, 84, 85, 135, 136, 138, Cabaj J...... 66, 116 139 Ceglarek P...... 35 Indyk D...... 99 Choromanska A...... 60 Jabłoński S...... 126 Chwiećko A...... 114 Jachimska B...... 45 Chwiłkowska A...... 122 Jakubczyk K...... 87 Cieniuch G...... 61 Jakuszko K...... 152 Cierluk K...... 62 Janda K...... 87 Cwynar P...... 63 Janił J...... 87 Cyboran-Mikołajczyk S...... 125, 160 Janiszewski T...... 77, 95 Czarnecka M...... 70 Jarmołkiewicz A...... 78 Czyż A...... 147 Jarynowski A...... 79

187 Jeżewska J...... 80, 153, 167 Krajewski A...... 97 Jęśkowiak I...... 81 Krajnik A...... 105 Józefiak H...... 151 Kratz E. M...... 96, 161 Junka A...... 68, 141 Kratz M...... 83 Jurczyk M...... 82 Krawczyk K...... 106, 107, 108 Jurkowska K...... 83 Krawczyk M. S...... 109 Juszczyk Z...... 106, 107, 108 Król M...... 110, 147 Jutel M...... 37 Krzywonos-Zawadzka A...... 111, 112, 133 Kaczmarek M...... 181 Krzyżak E...... 169 Kaim U. .. 43, 59, 75, 76, 84, 85, 135, 136, 138, Kubis-Kubiak A...... 67 139 Kulbacka J.32, 40, 42, 46, 60, 62, 65, 69, 90, 91, Kaisermann D...... 77, 95 132, 148, 163, 176 Kalinowska M...... 86 Kuliczkowski W...... 49 Kałduńska J...... 87, 92, 97, 142, 157, 166, 185 Kulus M...... 64 Kapczuk P...... 88, 113, 124, 155, 166 Kupaj B...... 103, 104 Karczewska J...... 48 Kupnicka P. ... 88, 113, 124, 155, 157, 166, 185 Karłowicz-Bodalska K...... 101 Kuropka P...... 52 Karolewicz B...... 93 Kuś P...... 114 Karpińska M...... 154 Kuzajewska D...... 123 Kasperkiewicz P...... 77, 95 Kuzan A...... 99 Kawalec A...... 89 Kwiatkowski S...... 32, 46, 90, 91 Kątnik E...... 31 Kwiecień A...... 115 Kepinska M...... 110 Lesiak A...... 116 Kicia M...... 152 Leśków A...... 52 Kielar M...... 56 Lewandowski Ł...... 38, 110 Kielbik A...... 32 Lewoń D...... 117 Kiełbasa E...... 94 Lian C...... 33 Kiełbik A...... 46, 90, 91 Lis-Kuberka J...... 118 Kikut J...... 87, 92, 97, 142, 157, 166, 181, 185 Liuta M...... 165 Kiszkiel J...... 44 Lizoń K...... 149 Klausa E...... 131 Lochyński S...... 47 Kleszczyńska H...... 160, 180 Lupa D...... 34 Kłak J...... 93 Łukasik Z. M...... 119 Kłosiński T...... 94 Łukaszewicz M...... 126 Knura M...... 178 Łukomska A...... 185 Kokot I...... 96 Maciejewska D...... 92, 97, 142, 157, 181 Kołodziejczyk K...... 51, 184 Majerz I...... 41, 109, 115 Kołodziejczyk-Czepas J...... 121 Majewska-Skórka M...... 181 Kołt S...... 77, 95 Makowska J. S...... 119 Kominek F...... 114, 145, 164 Małodobra-Mazur M...... 55 Komorek P...... 45 Małolepsza-Jarmołowska K...... 159 Komorniak N. .. 87, 92, 97, 113, 142, 157, 166, Maniewska J...... 120 181, 185 Marchewka D...... 122 Konias A...... 172 Marciniak K...... 103, 104 Kopacz Ż...... 152 Marczuk P...... 121 Korzeniowska-Kowal A...... 61 Markowska A...... 44 Kotas M...... 98 Markowska M...... 97 Kotowska A...... 99 Maruszewska A...... 123 Kotowski K...... 32, 46, 90, 91 Matczyszyn K...... 29, 33 Kotulska M...... 40 Matyniak D...... 92, 181 Kotyra Ł...... 100 Mazur M...... 180, 183 Kowalczyk A...... 58, 101 Mączyński M...... 81 Kowalewicz-Kulbat M...... 106, 107, 108 Metryka E...... 88, 97, 113, 124, 155, 157, 166 Kowalska K...... 102 Męcik-Kronenberg T...... 35, 128, 143 Kowalska P...... 103, 104 Męczarska K...... 125 188 Michel O...... 32 Piorun K...... 97 Mielko K. A...... 126 Piotrowska A...... 31 Mieszała K...... 160 Piwowar A...... 67, 72, 83, 96 Milewicz M...... 127 Płaczkowska S...... 96, 182 Milnerowicz H...... 38, 82, 102, 110, 117, 137 Podhorodecki A...... 116 Miłek O...... 128 Polesiak M...... 41 Młynarczykowska P...... 129 Popik M...... 92, 181 Młynarska K...... 177 Potyrak K...... 144 Młynarz P...... 126 Poznar M...... 154 Moczała M...... 154 Przybycień P...... 146 Moreira H...... 130, 175, 177 Przybyło M...... 36 Morka K...... 61, 131 Przybyszewski O...... 147 Mruk H...... 181 Przystupski D...... 32, 42, 46, 90, 91 Myszczyszyn A...... 55 Puchrowicz K...... 150 Nartowski K. P...... 93 Radajewska A...... 147 Niedziela M...... 35 Rajtczak-Wielgomas K...... 99 Niezgoda N...... 70, 132, 150 Raudner I...... 103, 104 Niziołek P...... 168 Rembiałkowska N...... 129, 148, 176 Novickij V...... 46, 176 Rogalski K...... 149 Nowak M...... 93 Rorbach-Dolata A...... 72 Nowakowska K...... 99 Rossowska J...... 22, 60 Nowicka J...... 51 Rudnicki J...... 153 Obmińska-Mrukowicz B...... 180 Rusak A...... 51 Oćwieja M...... 34 Rybak Z...... 51, 184 Ogorzałek M...... 134 Rychlicka M...... 150 Okła E...... 106, 107, 108 Ryczan-Krawczyk R...... 172 Olejnik A...... 111, 112, 133 Ryglowska A...... 149 Oleksy M...... 68, 141 Ryng S...... 81 Olesiak-Bańska J...... 63, 73 Ryszka F...... 140 Olędzki R. 43, 59, 75, 76, 84, 85, 135, 138, 139 Saczko J...... 32, 46, 60, 65, 90, 91, 148 OlędzkiR...... 136 Salvesen G...... 77, 95 Ołdakowska M...... 137 Samuel I. D. W...... 33 Orczyk-Pawiłowicz M...... 118 Sands D...... 126 Orkusz A. 43, 59, 75, 76, 84, 85, 135, 136, 138, Sarecka-Hujar B...... 151 139 Šatkauskas S...... 24 Ostróżka-Cieślik A...... 140 Satta E...... 76 Osyczka A. M...... 35, 128, 143 Sawicka E...... 83, 103, 104, 134, 171 Oszmiański J...... 125 Sawicki G...... 111, 112 Ożyhar A...... 156 Schubert J...... 131 Pajączkowska M...... 51 Sell J...... 152 Paleczny J...... 68, 141 Sępek M...... 80, 153, 167 Palma J...... 87, 92, 97, 142, 157, 166, 181, 185 Sikora A...... 154 Pańczyszyn E...... 143 Simińska D...... 88, 113, 124, 155 Pawelska M...... 177 Skibiński P...... 71 Pawełka D...... 55 Skonieczna-Żydecka K...... 97, 142, 181 Pawlak A...... 180 Skorupska A...... 156 Pawlik K...... 33 Skórka M...... 142 Pawlik-Sobecka L...... 96 Skórka-Majewicz M...... 92, 97, 157, 166, 185 Peregrym K...... 144 Słowiak A...... 158 Persheyev S...... 33 Smoleński M...... 159 Piasny J...... 177 Snipas S...... 77, 95 Piątkowska E...... 145 Sokolik R...... 96 Piątkowska P...... 134 Solarska-Ściuk K...... 160 Pieniężna A...... 78 Sołkiewicz K...... 96, 161 Piksa M...... 33 Sondaj K...... 50 189 Souto E. B...... 70, 132 Tyliszczak M...... 98 Spanu M...... 75 Tylko G...... 35, 128, 143 Spętana J...... 162 Ukleja-Sokołowska N...... 48 Sroka A...... 115 Ussowicz M...... 172 Stachowska E...... 92, 97, 142, 181 Uścinowicz P...... 44 Staniowski T...... 64 Van Elst D...... 37 Stańco W...... 163 Wachowska A...... 170 Starzec A...... 114, 145, 164 Wakulik K...... 175, 177 Stasik A...... 94 Walasek E...... 170 Stępień E...... 36 Wałecka-Zacharska E...... 131 Strub D...... 47 Wastag M...... 178 Strugała P...... 165 Wawryka P...... 32, 46, 90, 91 Stryburski D...... 181 Wawrzyniak M...... 141 Styburski D.87, 92, 97, 124, 142, 155, 157, 166, Ważna M...... 178 185 Wesołowska O...... 57 Surman M...... 36 Wiatrak B...... 175, 177, 182 Surowiak A...... 47 Wiczew D...... 42 Surowiak P...... 60 Wietrzyk J...... 70, 81 Sybirna N...... 165 Wiglusz K...... 179 Sycz Z...... 80, 153, 167 Wiglusz R. J...... 52, 179 Synowiec K...... 171 Witak W...... 86 Szczęśniak-Sięga B...... 120 Włoch A...... 125, 180 Szczukowski Ł...... 144 Wojciechowski J...... 40 Szkatuła D...... 168, 169 Wojnicz D...... 167 Szlasa W...... 39, 46, 50, 178 Wolska A. .. 87, 92, 97, 142, 157, 166, 181, 185 Szmidt M...... 170 Wołosik K...... 44 Szulc N...... 40 Wróbel P...... 116 Szwed K...... 171 Wysoczańska-Klaczyńska A...... 172, 182 Szydłowska B...... 170 Wzorek A...... 61 Szyjka A...... 130 Yoshida K...... 33 Szymański S...... 41 Zacniewski R...... 48 Szymonowicz M...... 51, 184 Zagała A...... 183 Ściskalska M...... 102 Zając P...... 144 Ślęzak A...... 172, 182 Zakrzewski W...... 184 Śliwińska-Hill U...... 173 Zaremba-Czogalla M...... 74 Śliwińska-Mossoń M...... 82, 117 Zborowska A...... 144 Świtalska M...... 70 Zemelka-Wiącek M...... 37 Świtała M...... 174 Zendran I...... 50 Tarek M...... 23, 40 Zoglowek A...... 30 Tarnowska M...... 52 Zuziak P...... 98 Tądel K...... 175, 177 Żarczyńska A...... 50 Terpińska M...... 172 Żurek P...... 154 Tomczak A...... 171 Żwierełło W.87, 88, 92, 123, 142, 157, 166, 181, Trembecki Ł...... 82 185 Trynda J...... 81 Żwirełło W...... 97 Tunikowska J...... 176 Żywicka B...... 51

190