Department of Biotechnology

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1 Department of Biotechnology Project 1 Prof. dr hab. Antoni Banaś MWB UG I GUMed Department of Biotechnology Laboratory of Plant Biochemistry The activity and substrate specificity of acyl-coa:3-phosphoglycerol acyltransferase (GPAT) in developing seeds of selected oilseed plants Aktywność i specyficzność substratowa acylotransferaz typu GPAT (acyl-coa:3-fosfoglicerol) w rozwijających się nasionach wybranych roślin oleistych Acylotransferazy typu GPAT są pierwszymi enzymami tzw. szlaku Kenediego; klasycznego szlaku biosyntezy triacylogliceroli. Poznanie zmiam ich aktywności i specyficzności substratowej może przyczynic sie do lepszego zrozumienia akumulacji triacylogliceroli w nasionach roslin oleistych. Jak do tej pory w Pracowni badaliśmy pozostale dwa enzymy szlaku Kenediego: acylotransferazy typu LPAAT (acyl-coa:kwas lizofosfatydowy) i acylotransferazy typu DGAT (acyl-coa:diacyloglicerol). Rozpoczecie badań nad enzymami typu GPAT pozwoli nam na skompletowanie informacji dotyczacych zmian aktywnosci i specyficznosci substratowej wszytkich enzymów szlaku Kenediego w rozwijających sie nasionach wybranych roślinoleistych. Project 2 Prof. UG, dr hab. Aleksandra Królicka MWB UG I GUMed Department of Biotechnology Laboratory of Biologically Active Compounds Therapeutic application of secondary metabolites from plants of Iris genus. (praca finansowana z programu MNiSW Diamentowy Grant) Terapeutyczne zastosowanie metabolitów wtórnych roślin z rodzaju Iris. The project includes the investigation of biological activity of secondary metabolites produced in plants tissue of Iris genus from Iridaceae family. These plants contain valuable secondary metabolites: isoflavones, quinones, flavonoids and triterpens which all have biologically active properties. The project presupposes a detailed activity analysis of substances isolated from Iris tissue (from in vitro and in vivo cultures, transformed roots) for testing cytotoxic activity on human cancer cell lines: HeLa, MCF-7, HL-60 and human skin cell line HaCaT. Also the antimicrobial activity will be tested on human, multidrug resistant pathogens: Klebsiella pneumoniae, Escherichia coli, Enterococcus faecalis, Candida albicans including those forming biofilm: Staphylococcus aureus and Pseudomonas aeruginosa. The application potential of extracts from Iris plants with combination with silver nanoparticles (AgNPs) in fight against bacterial and fungal infections will be tested as well. In the project a microfluidic device will be designed. This device will allow performing a quick and precise analysis of antibacterial activity of whole extracts as well as extracts with combination with silver nanoparticles, which underlines an interdisciplinary character of this project.

2 Project 3 Prof. dr hab. Ewa Łojkowska MWB UG I GUMed, promotor pomocniczy dr Robert Czajkowski, MWB UG I GUMed Department of Biotechnology Laboratory of Plant Protection and Biotechnology Identification, characterization and phylogenetic comparison of genes specifically up-regulated in Dickeya solani during colonization of roots, stems and leaves of Solanum tuberosum L. and Solanum dulcamara L. Identyfikacja, charakterystyka i filogenetyczne porównanie genów specyficznie nadeksprymowanych w bakteriach Dickeya solani podczas kolonizacji korzeni, łodyg i liści Solanum tuberosum L. i Solanum dulcamara L. (praca finansowania z projektu NCN SONATA 6) This project aims to identify, characterize and phylogenetically compare the genes present in D. solani that are specifically induced (up-regulated) during colonization of roots, stems and leaves of primary plant host for D. solani Solanum tuberosum (potato) and secondary (alternative) plant host Solanum dulcamara (bittersweet nightshade). It is assumed that plant colonization by bacteria is governed by a tight genetic regulation in which particular genes are turn on and off during different colonization stages and depending on colonized tissues (leaves, stems or roots) and plant host (S. tuberosum or S. dulcamara). The S. dulcamara has been chosen as a model secondary plant host due to its relatively high genetic closeness to S. tuberosum and because there are reports describing isolation of Dickeya spp. bacteria from symptomless S. dulcamara plants under natural conditions. The expected results of the project will provide information on the colonization of plant primary and secondary host tissues, specifically roots, stems and leaves by pectinolytic bacteria. The results will allow a better understanding of the colonization of plants plant pathogenic. The results will expend the general knowledge on plant microbe interactions and will add additional information to the model systems already existing. Department of Molecular and Cellular Biotechnology Project 4 Prof. dr hab. Jarosław Marszałek MWB UG I GUMed Department of Molecular and Cellular Biology Laboratory of Evolutionary Biochemistry Hsp70 molecular chaperones in mitochondrial biogenesis of iron-sulfur clusters: functional analysis and evolution Białka opiekuńcze Hsp70 w mitochondrialnej biogenezie centrów żelazo-siarkowych: analiza funkcjonalna i ewolucja. Iron-sulfur clusters (FeS) are ancient prosthetic groups critical for function of proteins in many processes essential for life, including electron transfer, catalysis, regulation of gene expression and environmental sensing. Specialized protein machineries that facilitate FeS biogenesis are present in all domains of life. In mitochondria, which play a central role in FeS biogenesis in eukaryotic cells, a dedicated chaperone system consisting of a member of the Hsp70 molecular chaperone family and its J-protein co-chaperone partner are critical for the transfer of the cluster to a recipient apoprotein. Our research focuses both on functional and evolutionary analyses of involvement of molecular chaperones in FeS biogenesis. By combining these two complementary approaches we answer questions relevant to both bio-medical aspects of chaperones function and evolutionary mechanisms behind functional divergence of molecular chaperones originated by gene duplication. We search for PhD students interested in molecular biology, biochemistry, cell biology and molecular evolution. Beside regular PhD fellowships additional founds for PhD students are available from "Maestro" and "Mistrz" projects funded by NCN and FNP respectively.

3 Project 5 Prof. dr hab. Jarosław Marszałek MWB UG I GUMed Department of Molecular and Cellular Biology Laboratory of Evolutionary Biochemistry Hsp70 molecular chaperones in mitochondrial biogenesis of iron-sulfur clusters: functional analysis and evolution Białka opiekuńcze Hsp70 w mitochondrialnej biogenezie centrów żelazo-siarkowych: analiza funkcjonalna i ewolucja. Iron-sulfur clusters (FeS) are ancient prosthetic groups critical for function of proteins in many processes essential for life, including electron transfer, catalysis, regulation of gene expression and environmental sensing. Specialized protein machineries that facilitate FeS biogenesis are present in all domains of life. In mitochondria, which play a central role in FeS biogenesis in eukaryotic cells, a dedicated chaperone system consisting of a member of the Hsp70 molecular chaperone family and its J-protein co-chaperone partner are critical for the transfer of the cluster to a recipient apoprotein. Our research focuses both on functional and evolutionary analyses of involvement of molecular chaperones in FeS biogenesis. By combining these two complementary approaches we answer questions relevant to both bio-medical aspects of chaperones function and evolutionary mechanisms behind functional divergence of molecular chaperones originated by gene duplication. We search for PhD students interested in molecular biology, biochemistry, cell biology and molecular evolution. Beside regular PhD fellowships additional founds for PhD students are available from "Maestro" and "Mistrz" projects funded by NCN and FNP respectively. Project 6 Prof. UG, der hab. Jacek Piosik MWB UG I GUMed Department of Molecular and Cellular Biology Laboratory of Biophysics Polyphenols isolated from raspberries as interceptors of food-borne mutagens. Polifenole z malin jako związki przechwytujące mutageny obecne w żywności. According to statistical data raspberries are a popular ingredient of everyday diet in the USA and Europe. Consumption of fresh raspberry fruits is believed to have a beneficial relation to human health, and that is why they have received much interest by both consumers and the researchers. Anthocyanins and ellagitannins isolated from raspberries possess several biological activities, especially anticancer properties. The exact mechanism of this chemopreventive action of anthocyanins and ellagitannins has not been established yet. It is hypothesized that these polyphenols can act as an interceptor molecules, sequestering aromatic mutagen molecules in stacking (π-π) complexes, contributing in this way to the decrease in its bioavailability. The main aim of this project is to verify this hypothesis. Within the project we want to establish the protective role of anthocyanins and ellagotannins from raspberries against food-derived mutagens, knows as heterocyclic aromatic amines (HCAs). We intend to investigate whether compounds mentioned above interact directly by the formation of stacking heterocomplexes. Moreover, we plan to establish to which extent such interactions may influence biological activity of the mutagens. The results of the project will give a new insight into the mode of action of anthocyanins and ellagotannins from raspberies as chemopreventive agents.

4 Project 7 Prof. dr hab. Michał Obuchowski MWB UG I GUMed, kopromotor prof. UG, dr hab. Rajmund Kaźmierkiewicz, MWB UG I GUMed Laboratory of Molecular Bacteriology Construct a model of the GerA germination receptor Skonstruowanie modelu receptora kiełkowania GerA (praca finansowana z projektu NCN HARMONIA 6) The bacterial spore is a specific type of the cell designed to persist harsh environmental conditions. Beside of the resistance for various conditions the spore need to continuously monitored environment. To achieve this the spore is equipped with germination receptors which can initiate transformation of spore into vegetative cell. The GerA receptor is activated by presence L-alanine or L-valine in the environment. However, the molecular mechanisms of its action remain unknown. In a frame of this project we like to try unravel molecular mechanisms of action of GerA receptors based on the molecular biology and molecular modelling approach. This will allow us to identify the substrate-binding pocket region and the contact surfaces between the subunits of the receptor, which will serve as guidelines for the construction of the group of mutants. Analysis of spore behavior after introducing selected mutations into the B. subtilis cells should provide data sufficient to propose a model describing the functioning of the receptor GerA in B. subtilis spore. Project 8 Prof. dr hab. Michał Obuchowski MWB UG I GUMed Laboratory of Molecular Bacteriology Mechanism of action of the Gera germination receptor of B. subtilis spore Analiza mechanizmu działania receptora GerA przetrwalników B. subtilis (praca finansowana z projektu NCN HARMONIA 6) The bacterial spore is a specific type of the cell designed to persist harsh environmental conditions. Beside of the resistance for various conditions the spore need to continuously monitored environment. To achieve this the spore is equipped with germination receptors which can initiate transformation of spore into vegetative cell. The GerA receptor is activated by presence L-alanine or L-valine in the environment. However, the molecular mechanisms of its action remain unknown. In a frame of this project we like to try unravel molecular mechanisms of action of GerA receptors based on the molecular biology and molecular modelling approach. This will allow us to identify the substrate-binding pocket region and the contact surfaces between the subunits of the receptor, which will serve as guidelines for the construction of the group of mutants. Analysis of spore behavior after introducing selected mutations into the B. subtilis cells should provide data sufficient to propose a model describing the functioning of the receptor GerA in B. subtilis spore.

5 Project 9 Prof. dr hab. Andrzej C. Składanowski MWB UG I GUMed Laboratory of Molecular Enzymology Analyses of molecular mechanism of FGFR inhibitors-triggered changes in growth and aggressive behavior of cancer cells in vitro studies. Analiza molekularnego mechanizmu wpływu inhibitorów FGF-R na wzrost i agresywność komórek nowotworowych w badaniach in vitro. (praca finansowana z projektu NCBiR STRATEGMED) W projekcie będzie porównywana skuteczność i selektywność inhibitorów FGFR uzyskanych z firmy farmaceutycznej Celon Pharma wobec komórek pochodzących z płasko-nabłonkowego raka płuca, żołądka i pęcherza moczowego. Zastosowane linie komórkowe będą pochodzić z nowotworów o zróżnicowanej złośliwości oraz posiadających różne odmiany receptora FGF będące skutkiem zmian genetycznych i epigenetycznych. Zasadnicze badania będą wykonywane w kulturach 3D sprzyjających zachowaniu oryginalnego fenotypu a zatem symulujących natywne oddziaływania z macierzą zewnątrzkomórkową i międzykomórkowe w środowisku. Wzrost komórek wewnątrz struktur trójwymiarowych będzie rejestrowany w czasie z użyciem systemu wideomikroskopowego. Badania kinetyczne wzrostu i ruchliwości w różnych środowiskach będą przeprowadzanie w komorze Boydena i za pomocą testu zabliźniania rany. Wzrost zależny i niezależny od kontaktu z podłożem będzie oceniany w płynnej agarozie. Wstępne doświadczenia mają na celu taką selekcje linii komórkowych aby znaleźć odmiany o zróżnicowanej czułości na zastosowane inhibitory FGFR. Kolejne etapy mają na celu analizę mechanizmu wpływu tych inhibitorów. W tych liniach będą przeprowadzone szeroko zakrojone badania specyficznej sygnalizacji z udziałem FGFR. Przeprowadzone będę też równoległe testy z udziałem białek efektorowych innych receptorów zaangażowanych w progresję nowotworów. Przeprowadzone badania dostarczą szeregu istotnych informacji o dotychczas nieznanych oddziaływaniach zależnych od receptora FGF a stymulujących rozwój raka. Będą one częścią szeroko zakrojonych badań klinicznych i przedklinicznych mających na celu rozwój funkcjonalnych leków na pewna określoną grupę nowotworów. Project 10 Dr hab. Anna Żaczek, MWB UG I GUMed; promotor pomocniczy dr Anna Markiewicz, MWB UG I GUMed, Laboratory of Cell Biology Clinical and biological significance of gene amplification and extranuclear form of estrogen receptor in breast cancer. Znaczenie kliniczne i biologiczne amplifikacji genu i pozajądrowej formy receptora estrogenowego w raku piersi. The presence of nuclear form of estrogen receptor (ER) in breast cancer cells is one of the most important predictive factors for hormonal therapy (eg. tamoxifen). Interestingly, the amplification of estrogen receptor gene (ESR1) has been found in primary tumors with and without nuclear form of estrogen receptor protein and it was associated with poor prognosis. Given that gene amplification is one of the mechanisms leading to protein overproduction, it is possible that ESR1 amplification leads to overexpression of ER, which accumulates in the cytoplasm. Yet, cytoplasmic estrogen receptor is not analyzed in a standard histopathological examination and literature data on its clinical and biological significance are scarce. The goal of a PhD project is to investigate the relationship between ESR1 amplification and cytoplasmic ER protein level in primary tumors of breast cancer and to assess the impact of ER cytoplasmic form on the progression of breast cancer. The examined markers will be correlated with clinical and pathological data, and survival of breast cancer patients, what should allow for verification of their clinical significance. In vitro model will analyze the impact of cytoplasmic ER signaling on phenotype of breast cancer cells and the effect of its inhibition by tamoxifen.

6 Laboratory of Biopolymers Structure Project 11 Prof. UG, dr hab. Stanisław OłdziejMWB UG I GUMed, kopromotor prof. dr hab. Krzysztof Łukaszuk, INVICTA, MUG Laboratory of Biopolymers Structure Proteomic biomarkers of human embryo developmental potential in in-vitro fertilization culture media. Biomarkery do określania potencjału rozwojowego ludzkich embrionów wydzielane do podłóż stosowanych w zapłodnieniach in vitro. The aim of the project is to identified proteomic biomarkers secreted by human embryo after in-vitro fertilization procedure. Biomarkers will be used for prognosis of further embryo development. The first stage of the project will involve isolation and chemical identification of substances secreted by human embryo to culture media used in in-vitro fertilization procedure. Further identified compounds, their concentration, secretion kinetics will be correlated with after implantation embryo development. The main technique used in the project will be mass spectrometry coupled with high performance liquid chromatography. Project 12 Prof. UG, dr hab. Stanisław Ołdziej MWB UG I GUMed, promotor pomocniczy dr Wioletta Żmudzińska MWB UG I GUMed Laboratory of Biopolymers Structure Phosphate and pyrophosphate binding motifs in proteins structure and catalytic properties. Białkowe motywy wiążące fosforany i pirofosforany struktura i własności katalityczne. The aim of the project is to identified short peptide sequences which are able to form stable threedimensional structure and binds phosphates or phyrophosphates and eventually catalyze reaction of phyrophosphate hydrolysis. Research will involve i) bioinformatic analysis of protein sequences to identify polypeptide sequences which are involved in phosphate and phyrophosphate binding; ii) chemical peptide synthesis, purification and structure determination using NMR spectroscopy; iii) for selected peptide assessing their phosphate or phyrophospahte binding abilities using HPLC, NMR, CD spectroscopy and mass spectrometry.

7 Laboratory of Virus Molecular Biology Project 13 Prof. dr hab. Krystyna Bieńkowska-Szewczyk MWB UG I GUMed, promotor pomocniczy dr Andrea Lipińska MWB UG I GUMed Laboratory of Virus Molecular Biology Identification of cellular proteins involved in the activity of bovine herpesvirus 1-encoded) UL49.5/glycoprotein M complex. Identyfikacja białek komórkowych uczestniczących w mechanizmie działania kompleksu UL49.5/glikoproteina M bydlęcego herpeswirusa 1 (praca finansowana z projektu NCN SONATABIS 4) The general project objective is to search for cellular proteins involved in the molecular mechanism of activity of alphaherpesvirus-encoded gm/gn (UL49.5) proteins, which act as key immune evasion molecules during infection of several alphaherpesviruses. The research models will include bovine herpesvirus 1 (BHV- 1), and to a lesser extent, protein homologs of herpes simplex HSV-1 and varicella-zoster virus VZV. gn (UL49.5) represents the major immunomodulatory protein inhibiting the activity of cytotoxic T lymphocytes during infection of BHV-1 and related alphaherpesviruses. gn interferes with the antigen presentation pathway via MHC class I molecules by blocking the TAP transporter. Complex formation with viral glycoprotein M (gm) regulates the activity of gn. In HSV-1-infected cells gm is internalized from the cell membrane by endocytosis and at the same time it can bind and internalize some cellular proteins, including the host restriction factors which belong to the components of innate immunity, like tetherin/bst- 2/CD317. Despite long tract of research on gm/gn homologs, the molecular mechanism of its activity remains elusive. To understand how gn/gm complex works we will apply broad multidirectional analyses, aiming at: identification of cellular proteins involved in the degradation of TAP by lentivirus shrna library screen and mass spectrometry analysis, elucidation of gm/gn role in internalization of tetherin and the mechanisms controlling this process, finally finding if the activity of alphaherpesvirus gm/gn homologs is evolutionary conserved or pointing out differences.