BOOK OF ABSTRACT

Transcription

BOOK OF ABSTRACT

BOOK OF ABSTRACT
XV National Academic Seminar of Biotechnology Students
&
V International Conference of Biotechnology Students
Wrocław,22-24 November 2013
Editors:
Aleksandra Pietrzyk
Natalia Plewa
Kamil Maciąg
Cover design:
Natalia Plewa
Main Sponsor
Honorary Patronage
Organizers Commitee
Chabowska Karolina
Ćwikłowska Kamila
Dąbrowska Adrianna
Gajdzik Weronika
Grudzień Małgorzata
Guziński Arkadiusz
Jasińska Martyna
Klein Adrian
Kołacz Joanna
Kozimor Weronika
Kużaj Agnieszka
Kwiatkowski Sylwester
Łopatecka Justyna
Maciąg Kamil
Mierzwicka Joanna
Mucha Marta
Niedźwiecki Piotr
Olechnowicz Zofia
Oliwa Agata
Olszówka Monika
Pająk Joanna
Pałka Marta
Pietrzyk Ola
Plewa Natalia
Puzio Marcelina
Siwek Magda
Skawiński Michał
Szczudłowska Ewa
Szlachetko Jagoda
Szul Justyna
Szymczak Kasia
Tran Józef
Trzeciak Karolina
Wasielewska Joanna
Way Anna
Zarecki Patryk
Zielińska Ewa
University of Wroclaw
Marie Curie-Sklodowska University of Lublin
The John Paul II Catholic University of Lublin
BOOK OF ABSTRACT
XV National Academic Seminar of Biotechnology Students
&
V International Conference of Biotechnology Students
Wroclaw 2014
Editors:
Aleksandra Pietrzyk
Natalia Plewa
Kamil Maciąg
Cover design:
Natalia Plewa
ISBN 978-83-936882-3-4
Copyright© by Aleksandra Pietrzyk & Natalia Plewa & Kamil Maciąg 2014
Print: Academic Society of Biotechnology Students
ul. Gronostajowa 7, pok. C101
30-387 Cracow
Dear participants
Welcome to Wrocław and the XV National Academic Seminar of Biotechnology Students
& V International Conference of Biotechnology Students.
On behalf of the organization committee I would like to thank all of you for taking part in the
conference. A warm welcome is especially sent to our prelegents and active participants,
without whom this conference would not exist.
The book you are holding is a collection of all active participants’ contributions, containing both
oral presentations and poster sessions, meant to help you quickly ascertain the purpose and
main aim of the posters and presentations.
In the following we are publishing the abstracts as submitted by the authors. We are not the
source of potential mistakes and other oversights.
We are certain you will have a rewarding time at the XV OASSB & V ISCB.
Natalia Plewa
Vice – President of Scientific Association of Biotechnology Students “Przybysz”
University of Wrocław
XV National Academic Seminar of Biotechnology Students & V International Conference of Biotechnology Students
Content:
Phage display as a tool in metastatic breast cancer therapy ...................................................................... 13
The application of image techniques in analysis of activated sludge flocs from wastewater treatment
plant............................................................................................................................................................. 14
Antioxidant diet in prevention of cancer .................................................................................................... 15
Spider silk: the intelligent biomaterial of the future ................................................................................... 16
Differentiation of induced pluripotent stem cells (ipscs) towards dopaminergic neurons. ....................... 17
Preliminary expression analysis of llfy gene from the flowering autonomous pathway of lupinus luteus. 18
Impact of baicalin and baicalein on splenoctyes, isolated from wistar rats ............................................... 19
Genetic diversity among slected ocimum l. Species using inter simple sequence repeat markers ............ 20
Alkohol`s influence on the human body ..................................................................................................... 21
Nanotechnology – properties and applications .......................................................................................... 22
Analyzing expression level of selected microrna molecules in healthy people
versus sarcoidosis patients.......................................................................................................................... 23
Revolution in medicine – 3d printing body parts ........................................................................................ 24
Plants as bioreactos – opportunities and challenges .................................................................................. 25
How to help wounds to heal? ..................................................................................................................... 26
Natural anti-cancer drug ............................................................................................................................. 27
Nongenomic mechanisms of 17β-estradiol and genistein effect on oral epithelial cells and human gingival
fibroblasts .................................................................................................................................................... 28
Harder, better, stronger, faster – caffeine and taurine .............................................................................. 29
Pyruvic acid production from crude glycerol by yarrowia lipolytica wratislavia 1.31 strain ...................... 30
Preservatives in shampoos .......................................................................................................................... 31
Screening of actinomycetales strains for their flocculating activities ......................................................... 32
Evaluationofanticanceractivityofextractfromcoffee (coffea l.) Againstcolorectaladenocarcinomacells .... 33
Antifungal activity of n-iodoacetyl derivative of amphotericin b against candida albicans ....................... 34
Methods for the extraction of extracellular polymeric substances from biofilm ....................................... 35
Bioconversion of crude glycerol to 1,3-propanediol: the resistance of enterobacteriaceae
to metabolites ............................................................................................................................................. 36
Uses of photosensitive substances in treatment of coronary in-stent restenosis...................................... 37
Degradation of natural estrogen by the microscopic fungus metarhizium robertsii .................................. 38
Nanosilver – particles of the future? ........................................................................................................... 39
Antioxidant interactions of coffee and cinnamon as aromatic supplement in respect to pure chemical
compoundsidentified in experimental material.......................................................................................... 40
An improved method for prediction of mammalian fibrinogen genes using genomic sequences ............. 41
7
The activity of ethanol and heptan extracts obtained from mallow (alcea l.) Against human colon cancer
cell line ht29 in vitro tests ........................................................................................................................... 42
The effect of virginia mallow (sida hermaphrodita l. Rusby) leaf and seed extracts onsiha (human cell line)
in vitro.......................................................................................................................................................... 43
Identification of mobile genetic elements in plat genomes........................................................................ 44
Biodegradation of nonylphenol by metarhizium sp. ................................................................................... 45
MtDNA polymorphisms in pediatric leukemia patients .............................................................................. 46
Presence of the urease genes in pseudomonas aeruginosa clinical strains................................................ 47
222
rn in utility buildings ................................................................................................................................ 48
Analysis of ant-ccrl1 antibodiesin human sera using quantitative dot blot method .................................. 49
Identification and analysis of llgamyb cdna in yellow lupine (lupinus luteus l.) Variety taper ................... 50
The use of bacteriophages as diagnostic and therapeutic agents ............................................................. 51
Vitamin d side-chain modified analogs and their impact on acute myeloid leukemia cell lines ................ 52
In search of the perfect match – computational analysis of potential mirna target sites........................... 53
The importance of cell hydrophobicity pseudomonas aeruginosa in biofilm formation............................ 54
Comparison of anticancer activity of native and boiled coelomic fluid from dendrobaena veneta ........... 55
Innovative systems for in vitro cultures of animal cells .............................................................................. 56
The examination of filamentous fungi ability to synthesize ligninolytic enzymes and their potential
contribution to the elimination of technical nonylphenol, 4-tert-octylphenol and 4-cumylphenol .......... 57
Polymieric nanocapsules for controlled drug delivery ................................................................................ 58
An antarctic lipase lipg7 – a new tool in biochemical engineering ............................................................. 59
Antimicrobial properties of zno nanoparticles............................................................................................ 60
Innovative additives in paper production – unique properties.................................................................. 61
Generation of transcription activator-like effector nucleases (talens) targeting adam10gene ................. 62
Viruses- a weapon against a cancer ............................................................................................................ 63
Dermathophythes – enzymatic virulence factors ....................................................................................... 64
The importance of surface hydrophobicity of proteus mirabilis cells in their ability to form biofilm on
solid surface................................................................................................................................................. 65
Evaluation of antibiotic susceptibility of uropathogenic escherichia coli strains........................................ 66
In vitro evaluation of cytotoxic effect of polyelectrolyte nanoparticles ant their application as a drug
delivery system............................................................................................................................................ 67
Collagen – sources and the production methods ....................................................................................... 68
Stevia rebaudiana – sweetness in nature ................................................................................................... 69
Recombinant biopharmaceuticals – where do they come from? ............................................................... 70
RNA structure of the influenza a virus- chemical probing .......................................................................... 71
Molecular mechanism of gdf-15 and sdf-1- induced angiogenesis – non-canonical role nrf2 transcription
factor ........................................................................................................................................................... 72
8
Influence of candida albicans metabolites on actin rearrangement in hep2 cells ..................................... 73
Tet protein (ten-eleven translocation proteins) – newly discovered regulators of expression and their role
in cancerogenesis ........................................................................................................................................ 74
The analysis of changes in the content and its derivatives analysis in cucumber (cucumis sativus l.) Seeds
one year after osmoconditioning ................................................................................................................ 75
Influence of platinum nanoparticles on human primary keratinocytes ..................................................... 76
Lowered cholesterol level or decreased cx43 expression inhibits tgf-β1-induced smad signaling in human
bronchial fibroblasts from asthmatic patients ............................................................................................ 77
Survival rate of microorganisms isolated from staff rooms of the food production plant in presence of
essential oils ................................................................................................................................................ 78
Sos response repressor of paracoccus aminophilus may influence transcription of antisense regulatory
rna ............................................................................................................................................................... 79
Influence of alternative disinfectants on the survival rate of microorganisms isolated from technological
areas in food industry.................................................................................................................................. 80
Titanium dioxide-coated paper – antimicrobial efficacy............................................................................. 81
Bacterial chromids – essential extrachromosomal genetic elements......................................................... 82
Dependency between gene p53 and neoplasia .......................................................................................... 83
Structure and properties oflignocellulosic complex.................................................................................... 84
Light it up ..................................................................................................................................................... 85
Yeast biosensor of tnt.................................................................................................................................. 86
Biotechnological methods of reducing the influence of abiotic stress factors on plants ........................... 87
The arrangement of lutein and zeaxanthin in popc lipid bilayer ................................................................ 88
The use of bacteriophages against listeria monocytogenes in dairy industry ............................................ 89
MicroRNA in asthma.................................................................................................................................... 90
Production of propionic acid on different sources of carbon by propionibacterium spp. ......................... 91
What is in a microscope image? Convolution and deconvolution .............................................................. 92
Phospholipids modification in paecilomyces marquandii under alachlore exposure. ................................ 93
useful toxins ................................................................................................................................................ 94
Aplication of pectin in food industry ........................................................................................................... 95
Mlpa- method of identification ofgene mutations ..................................................................................... 96
Biodegradation of bisphenola a by filamentous fungi emericella corrugata and mucor sp. ...................... 97
Screening for microorganisms capabpe of bisphenol a degradation.......................................................... 98
Identification ofa lifestyle-determining chromid of paracoccus aminophilus jcm 7686
(alphaproteobacteria) ................................................................................................................................. 99
Investigating the cross link between torc2, lipid metabolism and dna damage response ....................... 100
Effects of mutation in cinr and cini genes of rhizobium leguminosarum biovar trifolii strain 24.2 .......... 101
9
Synthesis, structural characterization and biological studies of ruthenium complexes with 2hydroxymethylbenzimidazole ................................................................................................................... 102
Brainbow and clarity – new methods to revolutionize neuroscience....................................................... 103
Design of the shrna nucleotide sequence and leading thread prediction in gene therapy ...................... 104
Alterations of manganese and cadmium transport in huntington’s disease ............................................ 105
The influence of sodium chlorine on synthetic estrogens utilization by selected filamentous fungi ....... 106
Cytotoxicity of thiosugars in cancer cell lines ........................................................................................... 107
Expression and purification of thermostable dna polymerases pfu and taq ............................................ 108
Two-dimensional gel electrophoresis image analysis in differential gel-based proteomics .................... 109
Nanocellulose- polymer of the future ....................................................................................................... 110
Elicitation and the phenylopropanoids precursor feeding as tools for improving nutraceutical potential
oflens culinarissprouts............................................................................................................................... 111
Changing in membrane lipids ofpenicillium chrysogenum in the presence of tributyltin chloride -a
reactive oxygen species (ros) inducer ....................................................................................................... 112
Relevance of citrullination in protein functions and pathological conditions .......................................... 113
Microenvironmental regulation of tumor progression and metastasis .................................................... 114
Phenolic compounds in selected edible forest fruits and their antioxidant properties ........................... 115
Phenolic compounds in selected poisonous forest fruits ......................................................................... 116
A new method of identifying pathogens – lamp as cost-effective and rapid alternative for pcr ............ 117
Qualitative analysis of the products of microbial biodegradation of 4-n-nonylphenol using gc-ms ........ 118
Next-generation sequencing – the future of molecular biology ............................................................... 119
The novel inhibitor of thioredoxin- thioredoxin reductase system .......................................................... 120
Bcl-2 – hope in the battle against cancer? ................................................................................................ 121
Analysis the detoxification properties of products from the group "detoxpad" ...................................... 122
Potential aplications of immobilized lactose in dairy industries ............................................................... 123
Review of immobilization method for microorganism ............................................................................. 124
Nanomaterials for bioanalysis and biotechnology applications. .............................................................. 125
Mysterious cytotoxicity assessment of zinc oxide nanoparticles in cell lines hela, nih-3t3 and a-549 .... 126
The effect of selected dietary supplements on the activity of glycolytic enzymes................................... 127
Application of confocal microscopy in plant research .............................................................................. 128
Some doubts about the tetrazolium salts ................................................................................................. 129
Changes in dna methylation profile in maize under herbicide stress ....................................................... 130
The effect of osmotic pressure on erythritol production from glycerol by yarrowia lipolytica................ 131
Enzymatic hydrolisis of dextran prior lc-ms/ms detection in urine samples for anti-doping control....... 132
Cellulose as a new polymer for the immobilization by extrusion method ............................................... 133
The influence of nrf2 transcription factor and heme oxygenase-1 on osteoclastogenesis ...................... 134
10
Farmacogenetics of anesthetic and analgesic agents ............................................................................... 135
Histone deacetylase inhibitors as a new multifunctional anticancer drugs ............................................. 136
Modification of polysulfone ag/sio2 ......................................................................................................... 137
Microbial corrosion of metals ................................................................................................................... 138
Fruits of selected species of forest plants as a source of healthy antioxidant substances in the diet of
birds ........................................................................................................................................................... 139
Little domestic killers................................................................................................................................. 140
The effects of the exogenous cholesterol on myofibroblastic transitions human bronchial fibroblasts
derived from asthmatic patients ............................................................................................................... 141
Molecular mimikry in autoimmune diseases ............................................................................................ 142
Human breeders ........................................................................................................................................ 143
Obtaining of mutants of s. Cerevisiae and p. Stipitis karyoductant to intensify the production of ethanol
from pentoses ........................................................................................................................................... 144
The importance of biofilms in fungal infections caused by candida albicans. Treatment and diagnosis of
candidiasis ................................................................................................................................................. 145
Antibacterial effect of the co-administration of dendrimers and antibiotics ........................................... 146
Mushrooms are not so black as they are painted ..................................................................................... 147
Filoviridae haemorrhagic fever viruses ..................................................................................................... 148
Microbial degradation of n-heterocyclic compounds ............................................................................... 149
Embryonic diapause in domestic species with seasonal reproduction – time to solve the enigma ......... 150
Cross-linking approach in structural and interaction studies between yin-yang 1 and its molecular partner
tata-box binding protein ........................................................................................................................... 151
The activity of ethanol and heptan extracts obtained from chamomile (matricariachamomilla l.) Against
human colon cancer cell line ht29 in vitro tests........................................................................................ 152
What is hiding in organic chemistry laboratory? ...................................................................................... 153
Lipopolisaccharide structure of pathogenic escherichia coli strains ......................................................... 154
Identification of probiotics by deconvoluted atr-ftir spectra ................................................................... 155
Genetic structure and functions of the chromosome of the arctic psychrobacter sp. Strain ................... 156
Epsilon protein – an essential element in plasmid psm19035 stabilization ............................................. 157
Vitamin d side-chain modified analogs and their impact on acute myeloid leukemia cell lines .............. 158
Investigation of lymphatic endothelial cell death mechanism after pdt .................................................. 159
The influence of tumor derived exosomes in human regulatory t cells in ovarian cancer. Preliminary
report......................................................................................................................................................... 160
Spheroid-plug model: a new tool in cancer research ............................................................................... 161
Restoration of atg5 to atg5-deficient du-145 cells brings back autophagy, however it does not change
sensitivity to photophrin-mediated photodynamic therapy ..................................................................... 162
Looking for a cure to diabetes - lysophosphatidylcholine ........................................................................ 163
11
New use of old drug: valproic acid promotes rhabdomyosarcoma cell differentiation ........................... 164
Phage therapy – medical applications and perspectives .......................................................................... 165
Synthesis and biological activity of new glucosamine-6-phosphate synthase inhibitors. ........................ 166
Adenovirus vector with muscle-specific expression casetteand lamin a gene for gene therapy of
laminopathies ............................................................................................................................................ 167
Spice plants under heavy metal stress conditions .................................................................................... 168
Polish trichoderma strains - biological control agents with plant protection potential ........................... 169
The use of palmaria palmata algae in the active biomonitoring of the surface waters ........................... 170
Polish trichoderma strains - biological control agents with plant protection potential ........................... 171
Commercially available applications of selected biotechniques variety in the captive bred reptile industry
based on example of python regius (shaw 1802) color morphs ............................................................... 172
Dynamic instability events in microtubule overlaps in vitro ..................................................................... 173
Bioactive compounds in coffee beans and their beneficial effect on human health ............................... 174
Integrative methodology for the identification of antiviral drugs against influenza protein targets ....... 175
Tale-tfs as gene therapy tool. Applications in congenita dysceratosis treatment .................................... 176
Expression of ccr3 as a single basophil identification marker ................................................................... 177
What do we know about rheumatoid arthritis? ....................................................................................... 178
12
PHAGE DISPLAY AS A TOOL IN METASTATIC BREAST CANCER
THERAPY
Agier Justyna1, Dadura Karolina1
1
The Microbiologic Section of Student‟s Scientific Association of Biologists, University of Łódź
Department of Immunobiology of Bacteria, University of Łódź, Faculty of Biology and Environmental Protection
Chemotherapy is the most common method for treatment of women with metastatic breast
cancer. Many cytotoxic drugs such as doxorubicin, vinorelbine, gemcitabine have low efficiency
and, what is more, large part of patients show high resistance to the chemotherapeutic agents. An
important problem in chemotherapy is the fact that drugs have no selective toxicity to normal
cells, which often leads to damage of healthy tissues and reduces the effectiveness of the therapy
(3). It is very important to develop new therapeutic strategies based on the construction of drug
delivery systems targeted directly at the tumor cells.
The phage display method is a molecular technique, first described by GP Smith in 1985. In this
technique phages are used to synthesize polypeptides which are applied as ligands specifically
interacting with cellular receptors (1,3). DNA fragments encoding the required peptide or protein
are inserted into phage capside genes. This process results in the expression of the protein on the
virus surface (2). The obtained peptides may mediate drug delivery, inhibit angiogenesis due to
their anti-cancer properties, reduce metastasis and block enzymes important in the cancer
development (1,5). The use of bacteriophage T7 in order to develop an anti-cancer vaccine seems
to be the most probable (4).
References
1. Budynek P., Dąbrowska K., Skaradziński G., Górski A. 2010 Bacteriophages and cancer.
Archives of Microbiology 192:315–320
2. Fagbohun O.A., Bedi D., Grabchenki N.I., Deinnocentos P.A., Bird R.C., Petrenko V.A. 2012
Landscape phages and their fusion proteins targeted to breast cancer cells. Protein Engineering,
Design & Selection 25:271–283
3. Lu R-M, Chen M-S, Chang D-K, Chiu C-Y, Lin W-C, et al. 2013 Targeted drug delivery
systems mediated by a novel peptide in breast cancer therapy and imaging. PLoS ONE 8(6):
e66128
4. Pouyanfard S., Bamdad T., Hashemi H., Bandehpour M., Kazemi B. 2012 Induction of
protective anti-CTL epitope responses against HER-2-positive breast cancer based on multivalent
T7 phage nanoparticles. PLoS ONE 7(11):e49539
5. Zawadzka Z., Adamczyk M., Tybinka A., Łątka A., Dąbrowska K., Górski A. 2012
Biotechnologia bakteriofagów: wybrane zastosowania metody phage-display. Postępy farmacji 3-4
13
THE APPLICATION OF IMAGE TECHNIQUES IN ANALYSIS OF
ACTIVATED SLUDGE FLOCS FROM WASTEWATER TREATMENT
PLANT
Olga Andrzejczak
1
Politechnika Łódzka, Wydział Biotechnologii i Nauk o Żywności, Wydział Technologii Fermentacji i Mikrobiologii
The charakteritics of activated sludge flocs are very important in operation of wastewater
treatment plants. Nowadays, computer analysis techniques can be very useful in the analysis of
activated sludge systems.
Automatic and semi-automatic image analysis procedure can be helpful in the evaluation of
activated sludge flocs. Thanks this method we can to provide such negative phenomena as
excessive fragmentation of activated sludge flocs or swelling of the sludge.
Computer analysis techniques can be used to the estimation of biomass concentration, to the
determine of morphological parameters of activated sludge flocs, to the determine of the
contribution of filamentous bacteria and the contribution of bacteria accumulating polyphosphate
(PAO), and at last to the identification of protozoa and methazoa.
Filamentous bacteria play an important role in the operation of activated sludge. They are
responsible for phenomena such as filamentous bulking or foam forming.
Fig 1. Image analysis techniques in monitoring of activated sludge.
14
ANTIOXIDANT DIET IN PREVENTION OF CANCER
Artur Anisiewicz1, Paulina Cieśla1, Joanna Florczak1, Joanna Haliniak1, Karolina Okła1, Monika
Orzełowska1, Anna Wawruszak1, 2
1
Scientific Students Groups of Biotechnology ”Mikron”, Faculty of Biology and Biotechnology, Maria CurieSkłodowska University, 20-033 Lublin, Poland
2
Department of Biochemistry and Molecular Biology, Medical University of Lublin, 20-059 Lublin, Poland
Reactive oxygen species (ROS), known as a oxidants, play a key role in the fundamental
processes of the human body. In a situation of oxidative stress occurs imbalance between the rate
of formation ROS and antioxidant system. Free radicals are highly reactive and quickly reacts
with proteins, sugars, lipids and nucleic acids, leading to formation the next free radical products.
DNA damage induced by ROS generate mutations leading to neoplastic changes. A growing body
of scientific evidence indicates that the inclusion of nutrients with strong antioxidant properties
to the daily diet reduces the risk of developing certain types of cancers. Among the products of
the most potent anti-cancer properties are pomegranates, green tea, red wine, curcuma and goji
berries.
References:
1.Donejko M., Niczyporuk M., Anti-cancer properties epigallocatechin-gallate contained in
green tea. Postępy Higieny i Medycyny Doświadczalnej, 67 (2013), s. 26-34
2.Lingling S., Guang D., Lycium barbarum polysaccharide stimulates proliferation of MCF-7
cells by the ERK pathway. Life Sciences, 91 (2012), s. 353-357
3.Shukla Y.Singh R., Reseveratrol and cellular mechanisms of cancer prevention. Annals of the
New York Academy of Sciences, Vol. 1215 (2011), s. 1-8
4. Wilken R., Veena M.S., Curcumin: A review of anti-cancer properties and therapeutics activity
in head and neck squamous cell carcinoma. Molecular Cancer, 10 (2011), s. 1-19
15
SPIDER SILK: THE INTELLIGENT BIOMATERIAL OF THE FUTURE
Agata Jędroszkowiak1, Adrian Kondrat1, Mateusz Antoszewski1
1
Studenci IV roku biotechnologii, Uniwersytet Przyrodniczy w Poznaniu
The unique properties of spider silk fibers, such as strength, extensibility and also, what finds to
be more significant in molecular biology, biocompatibility and biodegradability, are the reason of
its intense development.
Spider produces a few types of silk fibers with different mechanical properties, each of them in
other spinneret. Silk proteins – spidroins are built, among others, by repetitive sequences, which
play a main role in the silks properties. We are going to present usage of this unique properties in
medical biotechnology and others.
There are two strategies of spider silk proteins production. Construction of synthetic silk genes
with repetitive cDNA of consensus sequences of natural proteinsis the most often method. It
gives the possibility for engineered spider silk proteins with certain features: physical and
functional.
Tissue scaffolds, drug delivery spheres and anticancer usage will be described as the one of the
most promising part of the spider silk research. Although modifications, for instance,
metalloproteinase responsive domain may be give promising usage in future. Protein production
process in BLR (DE3) bacteria, bioreactor BioFlo 415 will be also part of the paper.
16
DIFFERENTIATION OF INDUCED PLURIPOTENT STEM CELLS
(IPSCS) TOWARDS DOPAMINERGIC NEURONS.
Maciej Sułkowski1, Tomasz Adamus1, Bogna Badyra1, Paweł Konieczny1, Marcin Majka1
1
Department of Transplantation, Medical College Jagiellonian University, Wielicka 265, 30-663 Krakow, Poland.
Parkinson‟s disease (PD) is one of the most prevalent neurodegenerative disorders. Despite of
particular pathological changes in morphology of brain tissue and behavioral outcome, etiology
of this disease is still not known. Recently discovered mutations in specific genes have been
conclusively shown to cause PD, but unfortunately, this genetic background of disease applies to
small percentage of patients.
In 2006 Yamanaka for the first time established induced Pluripotent Stem cells (iPS) by
introducing exogenous reprogramming factors into adult somatic cells . As pluripotent iPS cells
can differentiate into almost every cell type and possess unlimited proliferative potential.
Thanks to their traits they can potentially found application in clinic but also as in vitro models
in basic studies. Possibility of generating patient-specific iPSCs followed by their differentiation
towards particular cells can serve as a model for investigation of background of idiopathic
diseases like PD. Moreover, such biological material, after genetical modifications, may one day
be used as autological transplant.
Here we present protocol for neuronal differentiation of iPS cells. Neuronal differentiation is a
multi-step procedure. Firstly, iPS cells cultured on feeder layer, were transferred to suspension
culture. In next step Neuronal Progenitor Cells (NPC) were selected and expanded. In final step
NPC cells were terminally differentiated into dopaminergic neurons. On each step cells were
characterized for expression of specific markers (Nestin, Tuj-1, TH, DAT, embryonic markers) on
the level of mRNA.
17
PRELIMINARY EXPRESSION ANALYSIS OF LlFY GENE FROM THE
FLOWERING AUTONOMOUS PATHWAY OF LUPINUS LUTEUS
Mariusz Banach1,2, Waldemar Wojciechowski1,2, Jacek Kęsy1,2, Paulina Glazińska1,2, Emilia
Wilmowicz1,2, Agata Kućko1,2, Katarzyna Marciniak1,2, Jan Kopcewicz1, Andrzej Tretyn1,2
1
Chair of Plant Physiology and Biotechnology, Faculty of Biology and Environment Protection, Nicolaus
Copernicus University, Toruń, Poland
2
Centre for Modern Interdisciplinary Technologies, Nicolaus Copernicus University, Toruń, Poland
banach.mb@gmail.com
The flowering time is one of the factors providing reproductive success. At least four genetically
defined pathways have been identified that control flowering: vernalization pathway, photoperiod
pathway, gibberellin or hormonal pathway and autonomous pathway. All pathways control of
flowering cooperate regulation of key flowering genes which name „integrator genes‟.
The autonomous pathway of flowering induction genes include at least 7 genes. All these genes
are negative regulators of FLOWERING LOCUS C (FLC) - main inhibitor of flowering
induction.
FLOWERING TIME CONTROL PROTEIN (FY) plays dual roles in FLC regulation. In likely
model, FY acts in conjunction with FCA to repress FLC, but also has an FLC-promoting activity
that is FCA independent.
FY is an RNA 3‟ end-processing factor. FY interacts with RNA BINDING / FLOWERING TIME
CONTROL PROTEIN FCA ALPHA (FCA) (FY/FCA) by two proline-rich (PPLPP) motifs in the
C-terminus of FY. FY/FCA interaction shares in the autoregulation of FCA expression and the
selection polyadenylation site in the FLC pre-mRNA.
In this study, expression of LlFY gene was quantitative used real time PCR technique. Lupinus
luteus were cultivated in the field (soil class RIII) in Grubno(N: 53° 20' 30" E: 18° 28' 31"). L.
luteus seeds were sowed onfivedatesin April and May. RNA was isolated from leaves, roots and
flowers which were collected in 69th-43th days of cultivation.Plants organs were collected one day
from plants had been sowed on five dates.
Preliminary results obtained here will enable us to determineLlFY expression pattern in
vegetative and generative organs of L. luteus cultivars – the agricultural valuable species in
Poland. Expression of LlFY gene was the highest in the oldest leaves but the lowest in the oldest
flowers. Expression of LlFY gene was dependent on type of plant organs and their age. It will
also facilitate to characterize the role of these genes in the regulation of development of L. luteus
crops.
Acknowledgements:
This research was supported by the Ministry of Agriculture and Rural Development (Poland)
grants No 149/2011, the National Science Centre (Poland) grants No 2011/01/B/NZ9/03819 and
Nicolaus Copernicus University grants No 1522-B
18
IMPACT OF BAICALIN AND BAICALEIN ON SPLENOCTYES,
ISOLATED FROM WISTAR RATS
Edyta Bańcyr1, Isaura Felcenloben1, Radosław Gniłka2, Edyta Kostrzewa-Susłow2
1
Wroclaw University of Environmental and Life Science, Faculty of Veterinary Medicine, Department of
Immunology, Pathophysiology and Veterinary Preventive Medicine. Norwida 31 Road, 50-375 Wroclaw, Poland
2
Wroclaw University of Environmental and Life Science, Faculty of Food Science, Department of Chemistry,
Norwida 25 Road, 50-375 Wrocław, Poland
Baicalin and baicalein belong to a group of flavonoid compounds, isolated from roots and leaves
of Scutellaria baicalensis.Baicalin and baicalein have antioxidant activity, anti-angiogenic, anticancer, anxiolytic, anti-inflammatory, anti-viral, and they can stimulate production and
proliferation of human cytokines (Li et all 2000, Kitamura et all. 1998) Confirmed the successful
targeting of flavonoids extracts in the treatment of many diseases such as asthma, atopic
dermatitis, AIDS and liver cancer(Kubo et all. 1984, Huang et all 1986).
The aim of study: Affects of baicalin and baicalein for proliferation and survival of splenocytes,
isolated form Wistar rats
Material and methods: Isolated splenocytes from rats spleen‟s; proliferation of cells with different
concentration of baicalin or baicalein; survival of cells measure by Trypan Blue, phenotype of
cells checked by flow cytometry, proliferation checked by MTT test .
Results. Baicalin and baicalein changed proliferation of cells isolated form rat‟s spleen.
Moreover, baicalin and baicalein have important impact for proliferation.
19
GENETIC DIVERSITY AMONG SLECTED OCIMUM L. SPECIES USING
INTER SIMPLE SEQUENCE REPEAT MARKERS
Katarzyna Barańska1, Iwona Jędrzejczyk2
1
Scientific Association of Biotechnology Students „Biox‟
2
Department of Plant Genetics, Physiology and Biotechnology University of Technology and Life
Sciences,Kaliskiego Ave. 7, 85-796 Bydgoszcz, Poland
The genusOcimum L., a member of Lamiaceace family, includes many genotypes which are
cultivated for their remarkable essential oil. In view of rich composition of essential oil (a large
quantities of tannins, saponins, flavonoids and polyphenols) Ocimum L. species are used as herbs,
spices, raw material for the production of perfumes and herbal toiletries, biopharmaceuticals,
tinctures and liqueurs. In holistic medicine extract from the leaves of basil is used for various
ailments, such as headaches, coughs, diarrhea and kidneys malfunction. Basil are also thought to
be a source e.g. antifungal, bactericidal, antimalarial, antispasmodic and stimulant substances.
Genetic diversity the genus Ocimum L. is poorly understood. Occurrence of polyploids, hybrids
and various chemotypes, cause misidentification of genotypes, what makes the breeding difficult,
as well as the conservation of genetic resources. For this reason, it‟s important to find a
appropriate marker system, which will allow to quick and precise genotypes identification. In
contrast to morphological traits, molecular markers are not prone to environmental influences and
accurately characterize the plants portraying the range of genetic relationship among the taxa. In
the present study, genetic diversity among selected Ocimum L. species was determined using
inter simple sequence repeat (ISSR) markers.
Our study confirmed previously reported genetic diversity among the Ocimum L. genotypes. We
suggest that ISSR analysis could help in identifying genetic variations among species of Ocimum
L. and may improve their breeding program, and also industrial and medicinal uses.
20
ALKOHOL`S INFLUENCE ON THE HUMAN BODY
Magdalena Bartosiak1, Anna Cieślak2, Katarzyna Dziubak3, Katarzyna Kawka4
1
Scientific Students Group of Biotechnology „Mikron‟, Faculty of Biology and Biotechnology, Maria CurieSkłodowska University, magda.bartosiak1@gmail.com
2
Scientific Students Group of Biotechnology „Mikron‟, Faculty of Biology and Biotechnology, Maria CurieSkłodowska University, annacieslak92@hotmail.com
3
Scientific Students Group of Biotechnology „Mikron‟, Faculty of Biology and Biotechnology, Maria CurieSkłodowska University,katarzynadziubak@wp.pl
4
Scientific Students Group of Biotechnology „Mikron‟, Faculty of Biology and Biotechnology, Maria CurieSkłodowska University,katarzynakawka007@gmail.com
Alcohol has a similar influence on a human organism as an anesthetic. Its potency depends on
many factors like concentration and the amount of alcohol, rate and frequency of consumption,
sort and quantity of food eaten during the consumption. The final effect also depends on
individual tolerance to alcohol that is age, sex and consumer‟s metabolism.
Alcohol‟s activity in human body can be divided into three phases: absorptive phase, peak phase
and post-absorptive phase. Alcohol is the most powerful during the first phase and the weakest at
the last one. The disruption level of psychophysical functions mostly depends on the amount of
the first dose and whether it was consumed with full or empty stomach. There are five basic
stages of alcohol‟s influence on an organism depending on blood alcohol concentration i.e
euphoria, lethargy, confusion, stupor and coma. The kind of alcoholic beverage and its
concentration determine the velocity of absorption. Beverages which contain 15-30% alcohol by
volume are absorbed very fast, while the weaker or stronger ones are absorbed much more
slowly.
Toxic influence of the alcohol on the organism depends on its content in blood. Blood alcohol
content (BAC) is commonly defined as a 100 mg of alcohol per 1 dl of blood. Lab blood tests
give the most effective results of BAC in an organism. Breathalysers which test the concenration
of alcohol in an exhalated air are less precise. Modern toxicology uses two different kinds of
methods of measuring BAC these are indirect methods which use exhalated air to measure blood
alcohol content or direct methods like enzymatic or gas chromatography methods which use
fluids to examine the content of alcohol in the blood. Indirect methods are not accurate enough in
comparision to direct ones. Moreover, direct methods are characterized by better sensibility, small
amount of required test material, simplicity and rapidity of tests. Drinking too much of any
alcoholic drink can have a number of undesireable short and long-term effects on a human body.
21
NANOTECHNOLOGY – PROPERTIES AND APPLICATIONS
Magdalena Bartosiak1, Anna Cieślak2, Katarzyna Dziubak3, Katarzyna Kawka4
1
Scientific Students Group of Biotechnology „Mikron‟, Faculty of Biology and Biotechnology, Maria CurieSkłodowska University, magda.bartosiak1@gmail.com
2
Scientific Students Group of Biotechnology „Mikron‟, Faculty of Biology and Biotechnology, Maria CurieSkłodowska University, annacieslak92@hotmail.com
3
Scientific Students Group of Biotechnology „Mikron‟, Faculty of Biology and Biotechnology, Maria CurieSkłodowska University,katarzynadziubak@wp.pl
4
Scientific Students Group of Biotechnology „Mikron‟, Faculty of Biology and Biotechnology, Maria CurieSkłodowska University,katarzynakawka007@gmail.com
Nanotechnology is a field of science which is being intensively researched and developed thanks
to the possibility of applying nano-sized objects in most fields of human activity and everyday
life.It is defined as the description and study of the behavior and operations on atomic-scale
objects, particles and macro-particles. In the interest of the nanoscience are those particular
operations which show that analyzed objects prove to have different properties than the same
objects analyzed in a greater scale. Nanotechnology deals with creating, examining, producing
and implementing of structures, devices and systems by controlling the size and shape of the
objects in a nanometric scale.
Nano-particles are objects which have no more than 100 nm in a single dimension. The bottom
border of the size of the nano-particles is basically the size of an atom, which is estimated at
approximately 0,2 nm. Nano-particles can have very different shapes: spheres, e.g. carbon
fullerenes, tubes and other various shapes, such as flakes or flower-resembling patterns.
The nano-materials constitute a broader issue than nano-particles. Those are ones that are built
from nanometric elements, which are 0,2-100 nm in size. Nanometric materials and particles
retain the physical and chemical properties that are typical for macro scale and furthermore they
possess a set of unique properties, which is connected with the size of the particles. This dualism
is one of the greatest advantages of such objects. Additionally, the size of nano-particles‟ allows
them, for instance, to interpenetrate through most barriers, also those on a bio-organic level. A
typical property of the nano-particles is also a far more developed specific surface area that of
traditional materials. Nano-materials compared to traditional ones boast many unique properties,
such as: increased toughness, sturdiness, creep resistance, sliding properties, higher biocompatibility of the nanometric biomaterials, higher chemical resistance and superhydrophilicity.
Chemical industry is the main market of the available nano-scale products. The semiconductors
are the most common used nano-products. Those products prove to be useful in chemical industry
(as chemical catalysts as well as water air and sewage purifiers), and also in electronics and other
branches of industry. The safety of nanotechnology usage (mostly the products of which) is a
subject of many discourses.
22
ANALYZING EXPRESSION LEVEL OF SELECTED MICRORNA
MOLECULES IN HEALTHY PEOPLE VERSUS SARCOIDOSIS
PATIENTS
Maciej Bąk1,3, Agnieszka Jaźwa1, Alicja Józkowicz1, Krzysztof Sładek2, Józef Dulak1
1
Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian
University, Krakow, Poland
2
2nd Department of Internal Medicine, Jagiellonian University School of Medicine, Kraków, Poland
3
Scientific Association of Biotechnology Students 'Mygen'
Sarcoidosis is a chronic, multi-organ, immune system disease of unknown etiology, characterized
by formation of non-necrotizing granulomas in different tissues leading to their damage or
malfunction. Because of the lack of unequivocally defined factor that induces the local
inflammation and increases activation of leukocytes it is believed to be an autoimmune disease
and its genesis involves impaired balance between leukocytes subpopulations on the molecular
level. This leads to uncontrolled cytokine release, activation of T cells and their polarisation into
Th1 phenotype as well as increased activity of T regulatory lymphocytes [1,2].
MicroRNAs are small, protein non-coding, regulatory RNA molecules that influence gene
expression within the cell on the level of translation. Despite the fact that reactions of immune
system are mostly controlled by transcriptional factors microRNA play a significant role in both
innate and adaptive immunity [3].
The aim of our study was to determine and compare the level of expression of selected
microRNAs in healthy people and sarcoidosis patients. We detected decreased expression of
miR-150 and increased levels of miR-155 in the serum of patients when compared to healthy
people. On the other hand, the level of expression of these molecules is comparable in peripheral
blood mononuclear cells (PBMC) isolated from both groups.
These encouraging preliminary data should be followed by a more detailed studies, which
hopefully will allow us to characterize disease‟s pathogenesis.
References:
[1] M. Bordignon, P. Rottoli, C. Agostini, and M. Alaibac, “Adaptive immune responses in
primary cutaneous sarcoidosis.,” Clinical & developmental immunology, vol. 2011, p. 235142,
Jan. 2011.
[2] A. O‟Garra and P. Vieira, “Regulatory T cells and mechanisms of immune system control.,”
Nature medicine, vol. 10, no. 8, pp. 801–5, Aug. 2004.
[3] E. Tsitsiou and M. a Lindsay, “microRNAs and the immune response.,” Current opinion in
pharmacology, vol. 9, no. 4, pp. 514–20, Aug. 2009.
23
REVOLUTION IN MEDICINE – 3D PRINTING BODY PARTS
Iwona Bełczącka1
1
Scientific Students Groups of Biotechnology ”Mikron”, Faculty of Biology and Biotechnology, Maria CurieSklodowska University, 20-033 Lublin, Poland
3D printing (additive manufacturing) is the one of the most revolutionary technology in recent
years. It is used in many fields, including medicine. Already, 98% of hearing aids is printed and
this technology is commonly used by dentists. What else can we expect from 3D printing? First
of all, the individual implants and prostheses, scaffolds for tissue culturing or printed "plaster".
What technology will bring in the future? Printed blood vessels, skin, ears, tissue for testing
drugs, and for a while organs for transplantation. Such a futuristic dream remains far from reality,
but labs and private companies have already taken the first careful steps by using 3D printing
technology to build pieces of organs. Above all, scientists are working now, and the results of
their work soon may completely change the way we think about medicine.
References:
1.Greco N.D., Curent Advances in Tissue Engineering for the urinary tract. Tissue Engineering
Research Trends (161-181); 2008
2.http://www.livescience.com/39885-3d-printing-to-deliver-organs.html
3.http://on3dprinting.com/2013/08/30/3d-printing-and-medical-applications-a-full-roundup/
4.Fisher J., Mikos A., Bronzino J., Tissue engineering. CRC Press Taylor&Francis Group; 2006
24
PLANTS AS BIOREACTOS – OPPORTUNITIES AND CHALLENGES
Paulina Bierwagen1,2, Jakub Reiter1,2, Wanda Biała1
1
Uniwersytet Przyrodniczy w Poznaniu, Wydział Rolnictwa i Bioinżynierii
2
Koło Naukowe Studentów Biotechnologii OPERON, Uniwersytet Przyrodniczy w Poznaniu
One of the fields of biotechnology products in plants is the manufacturing of a wide range of
biologically active substances. Modern biotechnology allows to increase the range of products
derived from plants. These organisms are natural producers of valuable substances, what is more,
they are an excellent system for the production of heterologous proteins and other compounds of
non-plant origin.
As a production system plants have many advantages, they are readily available, easy to cultivate,
renewable and cost-effective. Another important benefit of plant producing system is their safety
for a human being. In contrary to animals there is no possibility of transmitting diseases from
plants to humans. Furthermore, green factories do not raise ethical issues. It is worth noting that
the procedures associated with the transformation and regeneration of plants are constantly being
improved, the application is simplified and manuals are more available.
Plants selected as bioreactors should have ease in shaping their genotype to obtain desirable
traits. Many species can be exploited for molecular farming, with arabidopsis and tobacco being
the most widely used host plants to date. Also various species used as a food or feed crop have
been tested. These include corn, rice and wheat, alfalfa, potato, oilseed rape, pea, tomato and
soybean. For example: in cereal crop recombinant proteins can be directed to accumulate in
seeds, which are evolutionary specialized for storage and thus protect proteins from proteolytic
degradation. The basic procedures used to adaptation of plants to producing the bioactive
substances are: transformation by co-cultivation with Agrobacterium tumefacies, or transiently by
infiltration with transgenic agrobacteria or transfection with viral vectors. Besides the molecular
methods using to modulate the natural products profile, we can also exploit plants natural
response to stress factors. Apart from the proteins we can also receive substances like hormones,
antioxidant or vitamins. Plant cell cultures have not been used as frequently as intact plants for
molecular farming but there are several examples that demonstrate their suitability as hosts for
the production of recombinant proteins.
Although molecular farming still faces a number of challenges, one can clearly see the benefits
and possibilities of the technology. Further research is needed to meet these challenges and
facilitate the cost-effective and safe production of pharmaceuticals and nutraceuticals in plants.
25
HOW TO HELP WOUNDS TO HEAL?
Karolina Boguszewska1
1
Institute of Technical Biochemistry, Faculty of Biotechnology and Food Science, Lodz University of Technology,
Stefanowskiego 4/10, 90-924 Łódź, Poland, Scientific Assosiation of Biotechnology Students FERMENT
Due to the increasing number of people with diabetes in recent years, research is conducted
increasingly in order to determine the problems associated with it. Diabetes is a condition known
to impair the normal course of wound healing, thus leading to chronic wounds and nonhealing
diabetic ulcers which account for approximately 25-50% of all hospital admissions in the diabetic
population. According to The International Diabetes Federation data (2011) about 8% of Polish
population suffers from diabetes and it has been estimated that in 2030 this value will exceed
10%.
Wound healing typically occurs in four phases: hemostasis, inflammation, proliferation and tissue
remodeling. The moment the tissue is injured healing response commences. Along with spill of
blood components into the wound, the platelets come into contact with collagen and other
compounds. Consequently the platelets release clotting and growth factors. Following
hemostasis, inflammatory phase begins in order to remove damaged tissue, bacteria and foreign
elements from the wound. It is accomplished by neutrophils and macrophages in the process of
phagocytosis. Subsequently, fibroblasts migrate in to the injury site to begin the phase of
proliferation. The new collagen matrix is created. During remodeling phase, matrix becomes
cross-linked and organized. In order for this repair process to take place there are required
numerous cell-signaling events. However, orderly sequence of four wound healing phases
overlapping in time and space is impaired under certain pathophysiological conditions such as
diabetes mellitus, venous and arterial insufficiency, chronic glucocorticoid use, obesity, aging,
stress or addictions.
Recent studies have demonstrated that extracellular nucleotides take part in all phases of wound
repair and those effects are mediated through P2 nucleotide receptors. Howewer, despite
excelling understanding of impact of the nucleotides on the healing process, many problems
remain to be solved. Due to the future therapeutical applications of nucleotides the knowledge of
signaling pathways and other factors must be improved.
26
NATURAL ANTI-CANCER DRUG
Martyna Boryń1
1
Faculty of Biotechnology and Food Sciences, Institut of Technical Biochemistry, Łódź University of Technology,
Scientific Association of Biotechnology Students FERMENT
Onconase (ranpirnase) is a protein isolated from Rana pipiens eggs and early embryos,
homologous to pancreatic RNase A. The activity of onconase, and particularly its antitumor
effect, is strictly associated with ribonuclease activity. Ranpirnase influences protein synthesis by
degrading tRNA (prevents translation) and other nucleic acids (e.g. siRNA) which cause changes
in gene expression. There are numerous molecular targets for onconase, therefore its antitumor
activity is a sum of its influence on several important
pathways.
One site of therapeutic action of onconase is prevention of
cells proliferation causing tumor cells death Firstly, it
inhibits the expression of cyclin D3, secondly, increases
the activity of cyclin-dependent kinase inhibitor proteins.
This, in turn, results in a decrease of pRB protein
phosphorylation in cells and retain them in G1 phase of
cell cycle. “Normal” cells can retain in resting phase for a
very long time, in contrast to tumor cells, which lose
viability and undergo apoptosis.
Another antitumor effects of onconase are:
1. Reduction of the level of H2O2 ,which targets NFκB activity by interfering with its nuclear
translocation.
2. Inhibition of metalloproteinase secretion.
Interestingly, onconase does not require a presence of p53 protein to induce cell death, which is
relevant due to production of inactive p53 protein in tumor cells.
Ranpirnase is the first cytotoxic ribonuclease to enter clinical trials for the treatment of cancer.
27
NONGENOMIC MECHANISMS OF 17β-ESTRADIOL AND GENISTEIN
EFFECT ON ORAL EPITHELIAL CELLS AND HUMAN GINGIVAL
FIBROBLASTS
M. Chmielewska1, P. Urbaniak2, M. Jendraszak2, I. Skibińska2, M. Kotwicka2
1
Medical Biotechnology, Poznan University of Medical Sciences,
2
Department of Cell Biology, Poznan University of Medical Sciences
Estrogens belong to a group of steroid hormones, which have been shown to act in
multidirectional way. Estrogenic effects are mediated by two types of intracellular receptors:
estrogen receptor 1 (ESR1) and estrogen receptor 2 (ESR2). There are highlighted two basic
mechanisms of estrogen actions: 1) classical-genomic and 2) nongenomic one. The second one
had been observed in several cell types of different origin. It leads to distinct effects, depended on
tissue and ligand type. However, the exact mechanism is still not fully understood.
It is also assumed that phytoestrogens, such as genistein, using endogenous estrogen intracellular
pathway of signal transduction, have significant impact on human cells biology.
Sex hormones are also expected to play important role in periodontal tissue modulation in
different periods of life, in both women and men.
The purpose of this research was to estimate effects of 17β-estradiol and genistein on oral
epithelial cells and human gingival fibroblasts in in vitro conditions.
The model cell lines in our studies were SCC-25 human tongue carcinoma cells (ATCC®) and
human gingival fibroblasts (HGF, Innoprot®). Cells were stimulated with different 17β-estradiol
(Sigma Aldrich®) and genistein (Sigma Aldrich®) concentrations. The final dilutions were
calculated to: 10-4 mol/l, 10-6mol/l, 10-8mol/l and 10-10 mol/l. After 12, 24, 48 and 72 hours of
incubation, cells morphology and proliferation were evaluated (Cell Index value was estimated
using xCELLigence system, Roche®). Changes of intracellular calcium ions concentration were
analyzed after incubation with fluorescent calcium indicator (Fluo-3, Invitrogen®), under
confocal microscope LSM 510 (Zeiss®).
Both of the analyzed hormones did not affect SCC-25 and HGF cells morphology. Analyzed
ligands did not alter SCC-25 cells proliferation in a significant way. No changes of intracellular
calcium ions concentration were observed, irrespective of hormone type and its dose. Both, 17βestradiol and genistein stimulated HGF cells proliferation in a statistically significant way,
depending on hormone type and its dose. 17β-estradiol stimulated rapid (observed after few
seconds), transient, dose dependent increase of calcium ions in human gingival fibroblasts.
In conclusion, 17β-estradiol and genistein affect human gingival fibroblasts in nongenomic
manner, through different mechanisms of action.
28
HARDER, BETTER, STRONGER, FASTER – CAFFEINE AND TAURINE
Michał Chojnacki1, Mateusz Pięt1, Artur Pachla1, Adrian Zając1
1
Scientific Students Group of Biotechnology „Mikron‟, Faculty of Biology and Biotechnology, Maria CurieSkłodowska University, mkpcho@gmail.com
Coffee is a beverage drunkall over the world. Some people can‟t imagine life without it. But what
are its effects on human organism? The authors will try to introduce coffee‟s main component,
caffeine, and a substance often used with pair with it, taurine: their structure, effects on human
organism‟s particular systems and the compounds‟ mutual interactions.
Caffeine is the main component of coffee. It is a purine alkaloid, contained naturally in leaves of
the tea shrub in coffee and cocoa beans as well as in other species of plants. Caffeine is almost
completely absorbed in the gastrointestinal tract and its maximum concentration in blood is
observed after 30-60 minutes after dosing. It easily penetrates the blood-brain barrier. It does not
accumulatein the body; its biological half-life is 3-4h.Caffeine is one of the most widespread
psychoactive substances in the world. Itstimulates thecentral nervous system andcancause a
variety ofeffectsonthe organism.Typically,it increasesmetabolic rate, heart rate,rate ofurine
production. It can alsoincrease productivity, improve themood,help relieve headaches, improve
alertness and reduce fatigue.Effects of caffeine on the human body depend on the dose. Caffeine,
first adopted in the amount of 100-600 mg, accelerates the course of thought and improves the
functioning of the body, but the doses above 2000 mg may cause insomnia, tremor, abnormal
cardiovascular system, impaired motor coordination and tachypnea. Caffeine stimulates
excluding the inhibitory mechanisms of neurons, mainly as a result of blocking adenosine
receptors in the brain. Caffeine by inhibition of adenosine receptors reduces glucose uptake by
muscles, which causes an increase of sugar in blood. Some publications suggest that caffeine has
antioxidant activity, but the mechanism of this phenomenon is not completely understood.
Taurine, although it is not contained in coffee, is often used in pair with caffeine, for example in
energy drinks; it may be also found in nutrients for sportsmens. The taurine belongs to amino
acids group. It is derivative of cysteine, but it contains sulfonate group instead of the carboxyl
group. Taurine participates in various metabolic processes responsible for the proper functioning
of the heart and skeletal muscles. It also reduces the brain serotonin production. The serotonin is
the inhibitory neurotransmitter of central nervous system and it reduces psychomotor activity. It
is also involved in bile acids synthesis, creatine transportation to muscles. Taurine may prevent
fatigue and extend physical and mental working capacity. In combination with caffeine it allows a
longer stimulatory.
29
PYRUVIC ACID PRODUCTION FROM CRUDE GLYCEROL BY
YARROWIA LIPOLYTICA WRATISLAVIA 1.31 STRAIN
Krzysztof Cybulski1, Magdalena Rakicka1, Ludwika Tomaszewska1
1
Department of Biotechnology and Food Microbiology, Wroclaw University of Environmental and Live Sciences, ul.
Chelmonskiego 37/41, 51-630 Wroclaw. E-mail: cybulski.krzysiek@gmail.com
Yarrowia lipolytica is currentlyone of the most extensively studied „„non-conventional‟‟ yeasts,
capable of producing a wide range of metabolites from cheap carbon source such as crude
glycerol. Nowadays pyruvic acidis produced mainly by chemical synthesis and has a number of
industrial applications. It is used as a building block in chemical synthesis of pharmaceuticals, as
a food additive, dietary supplement and in production of various polymers.
The aim of the present study was to evaluate the influence of pH, thiamine concentration and
nitrogen source on pyruvic acid biosynthesis by Yarrowia lipolytica Wratislavia 1.31 strain.
The studies were performed in 5-L stirred-tank bioreactor batch cultures (Biostat B+, Sartorius,
Germany) with working volume of 2 L at 30°C and the aeration rate fixed at 0.6 vvm. The stirrer
speed was set at 800 rev min-1. Different values of pH: 3.0, 3.5, 4.0, 4.5, 5.0 and 5.5 were
maintained automatically by the addition of 40 % (w/v) NaOH solution. An inoculum of 0.2 L
was introduced into a bioreactor containing 1.8 L of production medium. As the carbon source,
100 gL-1 crude glycerol was used. Source of nitrogen was ammonium sulfate, ammonium
chloride or urea. In the study thiamine concentration in the range from 0.4 to 4.0 μgL-1was tested.
Yeast produced from 25.2 to 45.4 gL-1 of pyruvic acid from 100 gL-1 of glycerol.pH value of 4.5
was found to be optimal for pyruvic acid production. At this value of pH Yarrowia lipolytica
Wratislavia 1.31 strain produced 35.3 gL-1 of pyruvic acid during 67 h cultivation. Thiamine
concentration was considered to be the crucial factor for pyruvic acid biosynthesis. The highest
amount of product (42.4 gL-1) was achieved when 1.5 μgL-1of thiamine wasapplied. The highest
pyruvic acid concentration was reached at pH 4.5 using urea as a nitrogen source, at thiamine
concentration of 1.5 μgL-1.Above mentioned batch culture was characterized by yield 0.45 gg-1
and volumetric acid production of 0.65 gL-1h-1.
30
PRESERVATIVES IN SHAMPOOS
Katarzyna Czapla1, Teresa Krzyśko-Łupicka2
1
Member of Students Scientific Association of Biotechnology,student of biotechnology
in cosmetics, Uniwersytet Opolski
2
Tutor of Students Scientific Association of Biotechnology,researcher, Uniwersytet Opolski
The goal of using preservatives is extending the microbiological stability of cosmetics. Depending on
the kind of product, there are used different preservatives. Ayurvedic cosmetics based on indian
medicine, may contain natural antimicrobial ingredients or chemicals, which are usually used in food
industry [1]. On the other hand, drugstore shampoos are mostly preserved with synthetic
compounds.The aim of the study was comparing the group of preservatives, used in ayurvedic and
drugstore shampoos. They are followed the composition given by producers (listed on the product
labels).
According to the producer‟s labels, the composition of four ayurvedic and four drugstore shampoos
with similar action was studied and compared.
The natural substances, having additional antimicrobial activity, are the major part of preservatives in
ayurvedic shampoos. These ingredients constitute an average of 11 ingredients per 16 of the total
constituent components. Chemical preservatives also appear in ayurvedic shampoos, but in a little
amount (0.5). However, in the composition of drugstore shampoos, there were an average of 5 chemical
preservatives per 24 of the total constituent components and 2 natural preservatives (see Fig. 1, 2).
Fig. 1. Ayurvedic shampoos
Fig. 2. Drugstore shampoos
The preservatives, used in ayurvedic shampoos, involve: vegetable extracts, essential oils and
chemical preservatives, which are used in the food industry, for example: potassium sorbate.
Moreover,drugstore shampoos include: chemical preservatives (often used in cosmetics such as
methylparaben), sodium benzoate, and much less frequently vegetable extracts, essential oils and
other natural compounds.
[1] Czapla K, Rost- Roszkowska M. Pielęgnacja skóry a ajurweda. Pol J Cosmetol 2011; 4; 245-251.
31
SCREENING OF ACTINOMYCETALES STRAINS FOR THEIR
FLOCCULATING ACTIVITIES
Magdalena Czemierska1, Aleksandra Szcześ2, Anna Jarosz-Wilkołazka1
1
Department of Biochemistry, 2Department ofInterfacial
Lublin, Poland
Phenomena, Maria Curie-Skłodowska University,
Bioflocculants are polymeric substances produced by various microorganisms during their
metabolism. They are mainly composed of proteins, polysaccharides, nucleic acids and
glycoproteins. These compounds are known as nontoxic, harmless and generally biodegradable.
For those characteristics, bioflocculants found various applications in industrial processes such as
wastewater treatment, heavy metals removal, fermentation and downstream processing.At
present, many scientists are searching for new microbial biopolymers with high flocculating
activity to improved already used applications in industry and to discover other potential fields
for their exploitation.
In this study, ten strains of flocculant-producing microorganisms were investigated. The
flocculating activity of all ten strains was tested and compared. The protein content was
determined by Bradford method and total sugar content was measured by the Phenol-sulfuric
method. Moreover, the surface morphology of bacterial cultures was observed by scanning
electron microscope (SEM).
This work was partially supported by the National Science Centre (2012/07/B/ST5/01799).
32
EVALUATIONOFANTICANCERACTIVITYOFEXTRACTFROMCOFFEE
(COFFEA L.) AGAINSTCOLORECTALADENOCARCINOMACELLS
Arkadiusz Czerwonka1, Maciej Frant1,2, Aleksandra Żurek1,2, Józef Kaczor1, Wojciech Rzeski1,3
1
Department of Virology and Immunology, Maria Curie-Skłodowska University, Lublin, Poland
2
Scientific Students Group of Biotechnology “Mikron”, Maria Curie-Skłodowska University, Lublin, Poland
3
Department of Medical Biology, Institute of Agricultural Medicine, Lublin, Poland
Coffee is one of the most commonly global consumed beverage (quantity of coffee consumed is
about 400 000 million cups every year [1] and second, after crude oil, worldwide commodity [2].
Most of commercial beans are produced by the two evergreen shrubs species Coffea arabica
(Arabica) and Coffea canephora (Robusta) cultivated in more than 60 subtropical and tropical
countries [3].
Consumption of coffee help prevent Parkinson's disease, liver disease, type 2 diabetes [4],
dementia and Alzheimer's disease [5] or even effect on stroke [6] and decreases body fat [7].
Furthermore, meta-analysis studies provides that coffee consumption had inverse association with
colorectal [8] pharynx, esophagus, kidney and some others types of cancers risk. [9]. However,
some of research reveals that, coffee consumption might be association with higher risk of
ovarian [10,11] or breast [12] cancer. Properties to increase or decrease the risk of developing
cancer appears to be related to non-caffeine components of coffee [13] and dose-dependent (four
or more cups of coffee per day are associated with a lower risk of endometrial cancer) [14] but
still in many case the relation between consumption of coffee and risk of cancer remains
unsettled.
One in four deaths in industrialized countries is due to cancer. The third most commonly
diagnosed and the second in terms of mortality is colorectal cancer [15]. There are several factors
associated with the development of colorectal cancer like diet high in animal fat and red meat
together with low in fruits and vegetables, environmental risk, low physical activity, obesity,
cigarette smoking and heavy alcohol consumption [16,17,18]. It is possible that multi-compounds
coffee brew rich in phenolic and melanoidins could be related to anti-carcinogenic effects in the
case colorectal cancer.
33
ANTIFUNGAL ACTIVITY OF N-IODOACETYL DERIVATIVE OF
AMPHOTERICIN B AGAINST CANDIDA ALBICANS
Aleksandra Czuryło1, Barbara Chudzik1, Daniel Kamiński2, Mariusz Gagoś1
1
Department of Cell Biology, Maria Curie-Skłodowska University, Lublin, Poland;
2
Department of Chemistry, University of Life Sciences, Lublin, Poland
Amphotericin B (AmB) is an antifungal antibioticused inhuman medicinefor over 50 years and is
considered the “gold standard” for the treatment of systemic fungal infections, especially in
people with weakened immune system. AmBis a valued antibiotic due to exceptionally high
antifungal activity and rare resistance among pathogenic strains. Despite many years of research
onAmB, the mechanism ofitscytotoxic activity is not fully understood. Many authors have
reported a greater affinityAmB to cell membranes containing ergosterol (fungal cells) compared
to the membranes with cholesterol (animal cells).
Still a problem is with the high toxicity of AmBin relation to the human cells, and therefore many
researchers is concentrated on the synthesis of the less toxic amphotericin B derivatives with high
antifungal activity. Equally importantis a better understanding of themechanism of action of AmB
which may contributeto the progress in the synthesis of the improved formula of the antibiotic.
The aim of our projectis to investigate the location of AmBin Candida albicans cell sand the way
of penetration across the cell wall. For this purpose, N-iodoacetyl derivative of Amphotericin B
(AmB-I) was synthesized,in which the iodo acetyl group is attached to the mycosamine of the
AmB molecule. Thanks to this modification it will be possible to find the intracellular location of
AmB moleculein C. albicans cells at molecular scale using electron microscope X-ray
fluorescence.
Preliminary studies showedthat N-iodoacetyl derivative of AmB has a slightly lower antifungal
activity against C. albicans than unmodified one.
Acknowledgement
This research was financed by the National Science Centre of Poland on the basis of decision no.
DEC-2012/05/B/NZ1/00037.
34
METHODS FOR THE EXTRACTION OF EXTRACELLULAR
POLYMERIC SUBSTANCES FROM BIOFILM
Dadura Karolina1,2, Agier Justyna 1,2, Moryl Magdalena2
1
2
The Microbiologic Section of Student‟s Scientific Association of Biologists, University of Łódź
Department of Immunobiology of Bacteria, University of Łódź, Faculty of Biology and Environmental Protection
Biofilm is composed of layers of microorganisms, which can communicate and interact with each
other. Bacterial contact with an abiotic surface could be a signal for extracellular polymers (EPS)
synthesis [1]. The EPS are composed of polysaccharides, proteins, phospholipids and the
extracellular DNA (eDNA) [3]. Polymers play an important role in bacteria adhesion to an abiotic
surface as well as in microcolony and mature biofilm formation. They also protect bacteria from
environmental stress. EPS functions also include: capturing and supplying nutrients to bacterial
cells, providing an optimum environment for genetic material exchange [2]
Isolation of the EPS and characterization of their structure and functions will contribute to a
better understanding of the processes occurring in the biofilm population. These studies could be
extended by developing new techniques (based on matrix degradation) for the bacterial biofilm
removal. The first step of our studies is biofilm cultivation on abiotic surfaces - plastic and glass
Petri dishes. The obtained bacterial biomass will be liophilizated and use as material for the EPS
isolation. EPS extraction methods can be divided into chemical and physical. Chemical
techniques include the use of 2% EDTA, formaldehyde and formaldehyde with NaOH. Physical
methods are based on rapid centrifugation and extraction with sonication [4].
References
1. Czaczyk K., Myszka K.: Biosynthesis of Extracellular Polymeric Substances (EPS) and Its
Role in Microbial Biofilm Formation. Polish J. of Environ. Stud. 2007; 6:799-806
2. Flemming H.C. et al.: The EPS matrix: The ”house of biofilm cells”.J Bacteriol. 2007;
189:7945–7947
3. Różalski A. et al.: Bakterie z rodzaju Proteus – cechy i czynniki chorobotwórczości. Postepy
Hig. Med. Dośw. (online). 2007; 61:204-219
4. Xiangliang P. et al: A comparison of five extraction methods for extracellular polymeric
substances (EPS) from biofilm by using three-dimensional excitation-emission matrix (3DEEM)
fluorescence spectroscopy. Water SA (online). 2010; 36:111-116
35
BIOCONVERSION OF CRUDE GLYCEROL TO 1,3-PROPANEDIOL:
THE RESISTANCE OF ENTEROBACTERIACEAE TO METABOLITES
Katarzyna Czaczyk1, Agnieszka Drożdżyńska1, Natalia Dąbrowska, Monika Drobna,
Ewa Dzwonkowska
1
Katedra Biotechnologii i Mikrobiologii Żywności, Uniwersytet Przyrodniczy w Poznaniu
1,3-propanediol (1,3-PD) is an organic compound widely applied in disparate branches of
industry. This molecule is mainly used as a monomer in reactions leading to synthesis
of polyesters, polyethers and polyurethanes. Due to combination of qualities such
as biodegradation, low toxicity and non-inflammability, 1,3-PD is successfully applied
in the production of medicines, cosmetics, detergents or plastics.
1,3-PD is primarily obtained by chemical methods, although to maintain the synthesis effective
the multistage processes require demanding production conditions. In search of alternative routes
biotechnological solution has been found. The use of microorganisms to produce 1,3-propanediol
results in less toxic by-products as well as lower costs. Furthermore, it creates the possibility to
employ the crude glycerol, deriving from the biodiesel production. The waste biomass, which is
still an unresolved issue, is normally utilized during bacteria metabolism.
The aim of the project was to test resistance of six strains of Citrobacter freundii and two strains
of Hafnia alvei bacteria to high concentration of glycerol, ethanol and 1,3-PD and screen them
for the ability to synthesize 1,3-PD most efficiently. The fermentation tests have been performed,
followed by qualitative and quantitative analysis of post-fermentation broth using high
performance liquid chromatography. The research resulted in selection of the three Citrobacter
freundii strains.
Biotechnological solutions in 1,3-propanediol production are remarkable progress in synthesis of
this compound. Finding natural producers of 1,3-PD will not only be more viable, but also
definitely less adverse from the ecological point of view. Considering the aforementioned
advantages, further studies on this subject might bring valuable results.
The paper was prepared within the framework of project no.01.01.02-00-074/09 co-funded
by The European Union from the European Regional Development Fund within the framework of
the Innovative Economy Operational Programme 2007-2013.
36
USES OF PHOTOSENSITIVE SUBSTANCES IN TREATMENT OF
CORONARY IN-STENT RESTENOSIS
Katarzyna Dudziak1, Justyna Bazan1,, Kinga Gostomska2, Anna Skotny3
1
Wroclaw Medical University, Laboratory of Neurotoxicology and Environmental Diagnostics, 51-618 Wroclaw, ul.
Bartla 5
2
Wroclaw University of Environmental And Life Sciences, 50-375 Wroclaw, ul. Norwida 25
3
Wroclaw Medical University, Department of Internal Diseases, Geriatrics and Allergology, 50-367 Wroclaw,
Wybrzeze L. Pasteura 4
The term restenosis mean a further reduction of coronary artery diameter after stenting.The risk
of this complication is closely related to the concentration of homocysteine in plasma. Numerous
studies have shown that restenosis forming inside the stent is caused by excessive growth of
neointimal. After stenting around foreign body starts a lot of reaction, including adhesion and
aggregation of platelets, which in turn leads to the formation of thrombus.
Photosensitizers after absorption of energy from an excitation light transfer their energy to the
tissue to produce reactive oxygen species (ROS, called reactive oxygen species). The area of
impact and transfer of singlet oxygen is 0.1 µm, which is more than sufficient to inhibit
endothelial cell division.
The result of photosensitizers actions is inhibition of the transmembrane transport of including
K+, Na+ and ATPase.Other cellular structures affected by changes are mitochondria, Golgi
apparatus, lysosomes and rough endoplasmic retlikum. In vitro studies confirm that the PDT
induces apoptosis or necrosis of endothelial cells. The effect is mediated by the release of
cytochrome c and caspase activation.
Excitation of photosensitizers besides antiproliferative activity by endothelial cell apoptosis, also
forms a barrier as a result of fibrosis of the intima cells of the vessel wall.This is caused by the
increased number of crosslinks of collagen in the extracellular matrix. It was also found that the
main reason for the inhibition of endothelial cell proliferation is not a denaturation of the binding
sites, but only the inactivation of fibroblast growth factor. Apoptosis focuses on the destruction
of neointimal.
References
1. Dudziak K, Master Thesis, Wrocław University of Technology 2012
2. SchnyderG, et al.The New England Journal of Medicine 2001; 345:1593-1600
3. MansfieldR, et al. Heart 2001;86:612–618
4. Wawrzyńska M, et al. Arch. Immunol. Ther. Exp. 2010 58:67–75
37
DEGRADATION OF NATURAL ESTROGEN BY THE MICROSCOPIC
FUNGUS METARHIZIUM ROBERTSII
Marta Dudzik1, Przemysław Bernat1, Paulina Siewiera1, Adrian Soboń1, Jerzy Długoński1
Department of Industrial Microbiology and Biotechnology, University of Łódź, Poland
Natural estrogens, e.g. estrone (E1), 17-β-estradiol (E2) and estriol (E3) are the most potent
endocrine disrupters. They are released by humans and livestock in the environment. The
conventional wastewater treatment is not efficient in the removal of natural estrogens. In spite of
the treatment, the effluent from conventional biological wastewater treatment systems still
contains estrogenic compounds at a level that may cause disruption of endocrine systems in some
species.
The aim of this study was to identify metabolic pathways that may be involved in the elimination
of estrogens and determination of optimal conditions for degradation of estrogen. To achieve this
aim, fungal strain Metarhizium robertsii IM 6519 was applied.
Quick, easy, cheap, effective, rugged and safe (QuEChERS) method was developed for sample
preparation. Then, a sensitive method for simultaneously determining free and conjugated
estrogens was developed using liquid chromatography–(electrospray) triple quadrupole–mass
spectrometry (LC–(ESI)MS–MS) in a negative mode.
The result shown that examined strain Metarhizium robertsii was able to grow in medium
containing 17-β-estradiol, however the estrogen partly inhibited fungal growth. After 5 days of
incubation, degradation of17-β-estradiol (at the initial concentration 10 mg/l) from samples was
almost completely. The main metabolites obtained in the fungal culture were estrone sulphate and
estradiol-17-sulphate. Additionally other metabolites, estradiol-17-glucuronide, estrone-3glucoronide, hydroxyestrone and estrone were found.
The QuEChERS method allows to obtain high quality results in analysis of estrogens residues in
medium. E2 metabolites produced in the fungus were identified and used to construct a proposed
estrogen transformation pathway.
This study was supported by the National Centre for Science in Krakow, Poland (Project No.
UMO-2011/01/B/NZ9/02898).
38
NANOSILVER – PARTICLES OF THE FUTURE?
Nina Dunajczyk, Joanna Dzierżawska
Lodz University of Technology, Faculty of Biotechnology and Food Sciences, ul. Wólczańska 171/173, 90-924 Łódź,
Poland Scientific Association of Biotechnology Students „Ferment‟
Nanosilveris amicroscopic particles-ionsof silver, which can be seen only by electron
microscope. Due to the fragmentation of silver nanoparticles with sizes ranging from1 to 5 nm
(read from 1 to 5 nanometers) is incomparably greater efficiency in the utilization bactericidal,
fungicidal and virucidal properties of silver. Reduced to silver nanoparticles is disproportionately
larger active surface and thus far unattainablebiocidal potential. The effectiveness of nanosilver in
clude elimination of over 99.99% of bacteria, fungi, virusesand mold.
The cell wall of the bacteria has a complex chemical composition (sugar- fatty- peptide), and one
of its ingredients is an amino acid- cysteine, which in its composition contains a reactive thiol
group (-SH).
There are several mechanisms of inactivation of bacteria :

Catalytic oxidation

Reaction of the bacterial cell wall

DNA Binding
Cosmetics containing nanosilver are recommended in the treatment of the following conditions:

mycosis of feet;

yeast infection;

acne;

bacterial infectionsof the skin;

staphylococcal infection;

infectionof the hair follicles;

seborrheic dermatitis;

non-allergiccontact dermatitis;

allergies;

damage, burns, cuts, wounds;

increased sweating;

polyps;

full bodyodorandfoot.
39
ANTIOXIDANT INTERACTIONS OF COFFEE AND CINNAMON AS
AROMATIC SUPPLEMENT IN RESPECT TO PURE CHEMICAL
COMPOUNDSIDENTIFIED IN EXPERIMENTAL MATERIAL
Agata Durak1, Urszula Gawlik-Dziki1
1
Uniwersytet Przyrodniczy w Lublinie, Wydział Nauk o Żywności i Biotechnologii, Katedra Biochemii i Chemii
Żywności
Despite the controversial effects of coffee on human health, coffee is nowadays accepted as a rich
source of compounds possessing antioxidant and radical scavenging activities and its commonly
consumed beverage in Europe and America.Spices and herbs havebeen added to foods since
ancient times, not onlyas flavoring agents, but also as folk medicines and food preservatives.
Presently, thereis an increasing interest both in the industry and inthe scientific research of spices
and aromatic herbs because of their strong antioxidant properties. It is worth noting, that many
herbs and spices, usually used to flavor dishes, contain phenolic compounds which have been
reported to show good antioxidant activity. Cinnamon used in this study as a coffee beverage
supplement, affect not only its flavor and aroma, but also antioxidant properties of coffee,
because this spice is a source of bioactive compoundssuch as cinnamic acid and coumarin.
To exert their biological properties, polyphenols have to be available to some extent in the target
tissue. Therefore, the biological properties of dietary polyphenols may depend on their absorption
in the gut and their bioavailability. The amount of bioaccessible food polyphenols may differ
quantitatively and qualitatively from polyphenols included in food databases. Moreover, most
studies on polyphenol bioavailability use mainly pure single molecules (isolated from food or
chemically synthesized) although their bioavailability from whole foods may be substantially
different.
The potential bioaccessibility and interactions between antiradical and anti-inflammatory
compounds from coffee and cinnamon were evaluated.The main phenolic acids of coffee
werecaffeoylquinic acid and its isomers, whereasfour proanthocyanidin, cinnamic acid and
coumarin, (Z)-cinnamaldehyde, (E)-cinnamaldehyde were identified in cinnamon extracts.Results
obtained for the whole plant material extracts were compared with those for chlorogenic and
cinnamic acids (main bioactive constituents of studied material). All samples, coffee, cinnamon
and their mixture showed abilities to scavenge free radicals:EC50values determined for antiradical
compounds averaged 1.86 mg/mLand 1.52 mg/mL for coffee and cinnamon extract,
respectively.Water-extractable coffee and cinnamon components showed similar lipoxygenase
(LOX) inhibitory activity: EC50= 4.22 mg/mL and 4.98 mg/mL for coffee and cinnamon extract,
respectively. Both activities increased after simulated gastrointestinal digestion. In the mixture
antiradical phytochemicals acted antagonistically - isoboles took the convex form. The same
interactions were determined for chemical standards. The water-extractable LOX inhibitors acted
synergistically – isobole curve was „concave‟. The same kind of interaction was determined for
standard compounds. Interestingly, after digestion in vitro slight antagonism in the action of LOX
inhibitors was observed. Obtained results show that food matrix and/or its changes during
gastrointestinal digestion may play an important role in creating the biological properties of
phytochemicals.
40
AN IMPROVED METHOD FOR PREDICTION OF MAMMALIAN
FIBRINOGEN GENES USING GENOMIC SEQUENCES
Maria Fornal1,3, Russel F. Doolittle2, Emanuel Smeds3
1
Department of Microbiology, Faculty of Biophysics, Biochemistry and Biotechnology, Jagiellonian University,
Krakow, Poland
2
Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, USA
3
Department of Clinical Sciences Lund, Division of Infection Medicine, Lund University, Lund, Sweden
Fibrinogen is a circulating glycoprotein that is responsible for forming fibrin upon cleavage by
the enzyme thrombin. It is a hexameric molecule with two A-, two B- and two -chains with a
total molecular weight of 340 kDa for human Fg. Since Fg is critical for blood clotting, it is a
fairly well studied molecule in humans and some laboratory animals, but is not well characterized
in many animals.
There are currently several mammalian genome-sequencing projects ongoing and this genomic
information may be used to predict a number of genes in silico, including the Fg genes. It has
previously been observed that thrombin and Fg are typically optimized for the same species, and
to be able to structurally understand this phenomena, an increased knowledge of the both
thrombin sequences and Fg sequences will be important. A better understanding of this system
may have important implications for testing new drugs affecting the coagulation system in
different animals during drug development. In addition to Fg‟s role in normal physiology, Fg also
seems to play a role in bacterial infections, in particular infections caused by gram-positive
bacteria.
Our initial analysis of the Fg genes for the best characterized species revealed that the commonly
used gene prediction software could not accurately predict the Fg genes. We set out to develop a
gene prediction method for mammals that could be used to predict Fg genes in mammals with a
better prediction rate than commonly used open source prediction software.
41
THEACTIVITY OF ETHANOL AND HEPTAN EXTRACTS OBTAINED
FROM MALLOW (ALCEA L.) AGAINST HUMAN COLON CANCER
CELL LINE HT29 IN VITRO TESTS
Maciej Frant1,2, Aleksandra Żurek1,2, Arkadiusz Czerwonka1, Roman Paduch1, Grażyna Matysik3
1
Department of Virology and Immunology, Maria Curie-Sklodowska University, Lublin, Poland
2
Scientific Students Group of Biotechnology “Mikron”, Maria Curie-Sklodowska University, Lublin, Poland
3
Department of Analytical Chemistry, Medical University, Lublin, Poland
Mallow (Alcea L.) is a plant from family Malvaceae. It grows in natural habitat in southwestern,
central and eastern Europe and southwestern Asia [1]. Marshmallow (Althaea officinalis – plant
from the same family) has been used in herbal medicine for many centuries. Mallow roots, in
order to their medicinal properties are commonly used in medicine, but the leaves and flowers of
this plant are also rich in many chemical compounds that have medical activity. In ancient times
boiled mallow root in combination with wine helped people to overcome numerous health
problems such as stomach upset, mallow combined with milk alleviated dry cough and mixed
with vinegar helped to combat toothache. Marshmallow is well known for its anti-inflammatory,
expectorant and emollient properties. It is used primarily in the respiratory tract inflammation,
due to the mucus contained in the plant, which is often used in the production of syrups
suppressants [2].
Scientific research of Iran‟s team on the Althea curdica(related species with mallow) on various
tumor lines in vitro have shown that mallow may have some anticancer activity [3].
Our study aimed to examine and compare the cytotoxic effect of extracts from mallow (ethanol
and heptanic) against tumor cells from colorectal cancer (HT29 cell line).For this purpose we
conducted two tests: NR uptake and MTT assay. The concentrations of the extracts were tested in
the range of 25 μg/ml to 250 μg/ml.
Different results was obtained for the two types of extracts. Ethanol extract showed a slight
cytotoxic effect (reduction of viability was only in the case of NR uptake – a 20% decrease
compared to control at the highest concentration), the heptanic extract showed a strong cytotoxic
activity to the HT29 cell line in both tests (decrease viability to 40% for NR uptake, and 14 % for
the MTT assay, at the highest concentration of the extract).
Bibliography:
Blamey M., Grey-Wilson C., The IllustratedFlora of Britain and Northern Europe, Lubrecht &
Cramer Ltd, 1989.
Williamson J., Wyandt C., Herbal therapies: The facts and the fiction, Drug topics, 1997, 78-87.
Khakdan Fatemeh, Piri Khosro, In vitro Cytotoxic Activity of Aqueous Root Extract of Althea
kurdica against Endothelial Human Bone Marrow Cells (line k562) and Human Lymphocytes,
Bulletin of Environment, Pharmacology and Life Sciences, 2013, 2(6): 23-29.
42
THE EFFECT OF VIRGINIA MALLOW (SIDA HERMAPHRODITA
L. RUSBY) LEAF AND SEED EXTRACTS ONSIHA (HUMAN CELL LINE)
IN VITRO
Maciej Frant1,3, Anna Kasprzyk2, Ewa Szczuka2, Roman Paduch1
1
Department of Virology and Immunology, Institute of Microbiology and Biotechnology UMCS, Akademicka 19, 20033 Lublin, Poland
2
Department of Plant Anatomy and Cytology, Institute of Biology and Biochemistry UMCS, Akademicka 19, 20-033
Lublin, Poland
3
Scientific Students Group of Biotechnology “Mikron”, Maria Curie-Sklodowska University, Lublin, Poland
Sida hermaphrodita belongs to the family Malvaceaeand it originates from southeastern parts of
Northern America.It was introduced to Poland in the 50s of the last century and it is interesting
because of potential economic importance: as biomass for energy generation, as a source of
fibers, in cellulose industry, as a forage and for planting in chemically degraded terrain. Virginia
mallow contains substances similar to medical comfrey and could be used in the pharmaceutical
industry.
The aim of the study was to investigate the effects of extracts activity of Sida hermaphrodita L.
Rusby (Malvaceae) against SiHa cell line. Extracts obtained from Virginia mallow leaves and
seeds were investigated for their anticancer effect. Two tests (MTT and NR uptake) were used to
test anti-proliferative activity and toxicity against SiHa (human cervicalcancer lines) in vitro.
Quite similar results were obtained for the two types of extracts. Leaves and seeds extracts
showed a slight cytotoxic effect (reduction of viability was only in the case of NR uptake – a
20% decrease compared to control), the both extractshave not shown anycytotoxic activity to the
SiHa cell line in MTT tests.
Bilbiography:
COSEWIC assessment and status report on the Virginia Mallow Sida hermaphrodita in Canada.
2010. Committee on the Status of Endangered Wildlife in Canada. Ottawa. ix + 18 pp.
Poińa L., Adamovičs A., Antipowa L., Ńiaudinis G., Karčauskiene D., Platače R., Ņukauskaite A.,
Malakauskaite S., Teirumnieka Ē. 2011. The chemical content of different energy crops.
Environment. Technology. Resources. Proceedings of the 8th International Scientific and
Practical Conference. Volume 1.
Kocoń A., Matyka M. 2012. Phytoextractive potential of Miscanthus giganteus and Sida
hermaphrodita growing under moderate pollution of soil with Zn and Pb. Journal of
Food,Agriculture & Environment Vol. 10 (2): 1253-1256.
43
IDENTIFICATION OF MOBILE GENETIC ELEMENTS IN PLAT
GENOMES
Seweryn Frasiński1, Tomasz Sakowicz1
1
Department of General Genetics, Plant Molecular Biology and Biotechnology, Faculty of Biology and
Environmental Protection, University of Lodz ul. Banacha, 12/16, 90-237 Lodz
Mobile genetic elements are a common component of all eukaryotic genomes. Until now they
have been identified in many animal, fungus and plant species. Most of them are an important
pool of genomic DNA sequences. They are extremely diverse in terms of their structure,
frequency, degree of autonomy, activity or mechanism of proliferation. Retroelements are one of
two major classes of mobile genetic element, created in genomes with the involvement of RNA
and reverse transcriptase. They are represented by large families of autonomous LINE elements
(Long Interspersed Nuclear Elements) and smaller, non-autonomous SINE elements. Both
groups of these families lack LTR (so called non-LTR), structures formed by long terminal
repeats characteristic of many other mobile elements. LINE-type sequences are involved in
amplification of other genetic elements, their activity can lead to genetic diseases. It is also
known that the reverse transcriptase of these elements plays a role in the development of some
cancers. Selected retrotransposons components are able to both stimulate and silence gene
expression. They are often used in research of so called genetic fingerprints.
There is relatively little progress in analysis of these sequences in plant and new information
about them can significantly enhance the current understanding of the mechanisms of genome
functioning. This justifies the interest in these sequences and undertaking research on LINE
elements in plants.
Identification and characterization of these sequences is carried out either by conventional
genetic engineering methods or by methods in silico. Significant progress in genome sequencing
projects causes that numerous, expanding databases specialize in collecting data concerning a
variety of repetitive sequences, which include LINE. Access to these databases as well as the
rich pool of specialized bioinformatic tools (including programs: NCBI BLAST, CENSOR,
ClustalX, Dotter, MrBayes, Emboss and many others) first allows finding new elements in the
genomic sequence, and then the multi-analysis (including comparative analysis). Among the beststudied plant species, also with respect to LINE sequences, are A.thaliana and O.sativa. Against
this background, the current knowledge of the corresponding elements in the family Fabaceae
(legume) is negligible. However, their ability to symbiotically fix atmospheric nitrogen makes
them an attractive object of study. Many of these species are of considerable economic
importance (soybean-an essential component of animal feed, as well as beans, peas or alfalfa).
44
BIODEGRADATION OF NONYLPHENOL BY METARHIZIUM SP.
Sandra Frączak1, Anna Różycka1, Adrian Soboń2, Sylwia Różalska2,
Jerzy Długoński2
1
Biotechnology and Microbiology Students Society “Bio-Mik” Department of Industrial Microbiology and
Biotechnology, University of Łódź, Poland
2
EDCs (Endocrine Disrupting Compounds) are manmade compounds that may interfere with the
body‟s endocrine system and produce adverse developmental, reproductive, neurological and
immune effects in both humans and wildlife. One of these compounds is nonylphenol. Species
belonging to M. anisopliae complex are present in the environment and are exposed to toxic
agents.
The aim of this work was the estimation of the 4-n-nonylphenol (4-n-NP) utilization ability in the
M. anisopliae complex.
Fungal cultures on Lobos medium were supplemented with 50 mg/l of 4-n-NP. After the
incubation, cultures were extracted with ethyl acetate and subjected to analysis on
GC-MS.
All species belonging to Metarhizium anisopliae complex have ability to utilize
4-n-NP. The most efficient species were M. velutinum, M. anisopliae, M. robertsii,
M. globosum and M. guizhouense, which remove about 95% of the 4-n-NP during 48h
of incubation. These species also differs in the dynamic of tested compound utilization.
M. robertsii and M. globosum are the best 4-n-NP degraders. The species
M. majus and M. lepidiotae had the weakest ability to remove this xenobiotic and at the end
of the incubation the amount of the 4-n-NP in the culture medium was 74 and 43%, respectively.
The 4-n-NP biodegradation product (4-hydroxybenzoic acid) was detected in all Metarhizium
cultures.
All species belonging to M. anisopliae complex have the ability to remove 4-n-NP from the
culture medium. Probably this is unique feature for the genus of Metarhizium.
45
MTDNA POLYMORPHISMS IN PEDIATRIC LEUKEMIA PATIENTS
Magda Ghanim1*, Agata Kodroń1, Katarzyna Jóźwiak1, Katarzyna Tońska1, Anna StelmaszczykEmmel2, Urszula Demkow2, Ewa Bartnik1,3
1
Institute of Genetics and Biotechnology, University of Warsaw, magdaghanim@yahoo.com
2
Department of Laboratory Diagnostics and Clinical Immunology of Developmental Age, Medical University of
Warsaw
3
Institute of Biochemistry and Biophysics, Polish Academy of Sciences
Mitochondria are responsible for energy production during cellular respiration. They contain their
own circular DNA of about 16568bp. mtDNA is more susceptible to damage than nuclear DNA
mainly due to less efficient DNA repair systems, the lack of protection of the genetic material by
histone proteins and mutagenic effects of free radicals generated in the process of oxidative
phosphorylation in mitochondria. So far, over 100 mtDNA point mutations causing human
disease have been detected, and more mutations are discovered every year. Numerousstudies
ofmtDNAin cancer have showndifferences in mtDNAsequencesbetweentumorand normaltissue
and at various stages ofcancer treatment in the same patient. However, there are few data on
acutelymphoblastic leukemia (ALL), the most common type of leukemia in children.
In this study we investigated mitochondrial sequence variation in the D-loop region and the ND1
gene (coding NADH dehydrogenase 1) in bone marrow cells and blood collected at the time of
ALL diagnosisandat various stagesof chemotherapy from 6 pediatric patients. We noticed the
presence of many rare polymorphisms in both control and coding regions of mtDNA samples of
all patients. In one patient we detected differences in the level of polymorphic variants at
positions 310, 16183 and 16189 in the D-loop region as well as at position 6680 in the coding
region of mtDNA from bone marrow cells derived from a patient before and after chemotherapy.
46
PRESENCE OF THE UREASE GENES IN PSEUDOMONAS AERUGINOSA
CLINICAL STRAINS
Dawid Gmiter1, Grzegorz Czerwonka1, Wiesław Kaca1
1
Department of Microbiology, Institute of Biology, The Jan Kochanowski University, Kielce, Poland
Pseudomonas aeruginosa is a one of the most import opportunistic pathogen related with cystic
fibrosis lungs infections. It is also observe that several studies describe this bacteria as an
ureolytic negative.
The aim of the study was to determine the presence of an urease subunits genes in P. aeruginosa
genomes. In the first step NCBI BLAST was used to compare known sequences of urease genes
with P. aeruginosa genomes. The alignment with pair of primers was done also. Next as a
confirmation the Polymerase Chain Reaction (PCR) technique was performed with collection of
43 P. aeruginosa clinical strains.
The investigation based on a bioinformatics analysis pointed out similarity between genomes of
ten P. aeruginosa strains and Proteus mirabilis urease operon as well as a Helicobacter pylori
ureB genes. Whereas, PCR with the assistance of an agarose gel electrophoresis demonstrated
presence of probability urease sequence in 42 strains.
This study showed that it is possible for P. aeruginosa to carry out urease gene. It was also
preamble to the further research focus on environment conditions which are necessary for urease
gene expression.
47
222
RN IN UTILITY BUILDINGS
Godyń P.1, Dołhańczuk-Śródka A.1, Ziembik Z.1, Kłos A.1
1
Independent Chair of Biotechnology and Molecular Biology, Opole University
Radon is one of the elements formed in the decay chain initiated by both uranium and thorium .
The radiation produced by radon decays exceeds 36 % of the annual effective dose of ionizing
radiation. This fact classifies radon as the most important source of radiation in environment.
222
Rn unlike the other radon isotopes such as 219Rn ( actinon ) , 220Rn ( thoron ) has a relative long
half-life - 3.8 day. The long half-life along with the physical properties of the isotope ( gaseous
state, density ) decide aboutriskslinkedwith exposure on 222Rn decays. 222Rn and its decay
products are one of the major causes of lung cancer. In the U.S. it is the second cause of this
disease, after smoking cigarettes.
222
Rn activity measurements were made using a portable spectrometer AlphaGUARD in two
buildings at the Opole University. The studies have shown greater exposure of people working in
rooms than people staying in the open spaces of the building. It was found that 222Rn specific
activity concentration was higher on the lower floors then on the upper one. However, studies
have not shown the potential risks to health from radon inhalation.
48
ANALYSIS OF ANT-CCRL1 ANTIBODIESIN HUMAN SERA USING
QUANTITATIVE DOT BLOT METHOD
Weronika Gonciarz3, Iwona Konieczna1, Inga Relich2, Beata Kolesińska2, Justyna Frączyk2,
Marek Kwinkowski1, Zbigniew Kamiński2, Wiesław Kaca1
1
Department of Microbiology, Institute of Biology, Jan Kochanowski University at Kielce
2
Institute of Organic Chemistry Technical University, Lodz
3
Institute of Immunology and Infectious Biology, Universityof Lodz
Introduction: In human sera are present antibodies which recognize specific antigens. In sera of
healthy people is varied range of antibodies. Simultaneously, their level is low. In sera of patients
with diagnosed systemic inflammatory diseases, especially with autoimmune background,
antibodies recognize specific foreign epitope, but due molecular mimicry, also protein with
similar structure and / or amino acid sequence. Atherosclerosis is classified as a chronic
inflammatory disease, which is the result of activation of innate immune system. However, its
autoimmune background is still discussed. In previous work, a similarity between Helicobacter
pylori urease and human C-C chemokine receptor type 11 (CCRL1) was observed.
Aim: The aim of this study was to compare the level of antibodies, in sera of atherosclerosis
patients and volunteer blood donors, recognizing synthetic peptides mimicking amino acid
sequence of extracellular domain of the human CCRL1 protein.
Materials and methods: Atherosclerosis patient (AP) (median age: 62±13), young volunteer blood
donor (yVBD) (median age 19±0.5) and old volunteer blood donor (oVBD) (median age 51±6)
sera were analyzed. Thirteen overlapping synthetic peptides mimicking sequence of extracellular
domain of CCRL1 were immobilized on cellulose membrane via triazine derivative and used as
an antigen for detection of the anti-CCRL1 antibodies. Antibodies level was determined by
quantitative dot blot method.
Results: The results shows, that in all analyzed human sera are present antibodies against
CCRL1, but their lever is differentiated depending on the group. Higher level of anti-CCRL1
antibodies was observed in AP sera. Sera from oVBD and yVBD showed similar level of
antibodies. Significantly stronger reactions of atherosclerosis patients sera, compared to blood
donors, were observed with mimetics corresponding to N-and C-terminal part of of CCRL1
extracellular domain. The level of antibodies recognizing synthetic peptides corresponding to the
central fragment of CCRL1 domain was similar in all groups of sera (patients and blood donors).
Conclusions: Atherosclerosis is a common disease. Increasingly, it is suggested that it is
autoimmune etiology. Characteristic feature of autoimmune diseases is the excessive reactivity of
the immune system, which produce antibodies, including autoantibodies, which may mediate the
formation and development of inflammation leading to atherosclerotic lesions. Presented results
confirm such suggestion.
Acknowledgements: This work was supported by grant UMO-2011/03/D/NZ6/03316 from the
National Research Center.
49
IDENTIFICATION AND ANALYSIS OF LlGAMyb cDNA IN YELLOW
LUPINE (LUPINUS LUTEUS L.) VARIETY TAPER
Katarzyna Marciniak1, Aneta Gorzelańczyk1, Mariusz Banach1
1
Uniwersytet Mikołaja Kopernika, Katedra Fizjologii Roślin i Biotechnologii, ul. Lwowska 1, 87-100 Toruń, Poland
GAMyb transcription factor is a positive component of gibberellin (GA) signal transduction
pathway andplays an important role in many aspects of plant growth and development, including
seed development, stem elongation andfloral initiation. A potential target for GAMyb in
regulation of flowering is LEAFY gene. All GAMyb proteins have characteristic DNA binding
domain (DBD) and three boxes with highly conserved sequences (BOX1, BOX2 and BOX3). At
present, GAMyb regulators are relatively well known inArabidopsis thaliana or cereals, but still
require detailed studies in other plant species, including legumes.
In our work,cDNA fragment of GAMyb gene in yellow lupine (Lupinus luteusL.) variety Taper
(epigonal form) has been identified. This result was obtained using RT-PCR method with
degenerate primers, designed based on the sequences of closely related species. Subsequently,
available on-line computer programs enabled translation of the nucleotide sequence (703 nt) to
the predicted amino acid sequence (234 aa). The presence of the Myb-like DBD and SANT
(SWI3, ADA2, N-CoR and TFIIIB' DNA-binding)domain indicate with a high probability that
this is a part of the functional enzyme protein. Recognized sequence exhibits also high degree of
identity and similarity to other plant species, which demonstrates its conservatism during the
evolution. Our results may suggest potential role of LlGAMyb protein in regulation such
processes as its homologues in different plant.
It should be added, that presented results are an introduction to the extensive research that in the
near future will determine the precise mechanism of yellow lupine flowering. This information is
very important because it is well known, that correctly running process of flower growth and
development determines the high productivity of legumes. In turn, strong development of
domestic production of grain legumes may be an alternative source of protein in Poland for
imported soybean.
This work was supported by the Ministry of Agriculture and Rural Development of Poland Grant
No 149/2011 and Nicolaus Copernicus University Grant No 1530-B.
50
THE USE OF BACTERIOPHAGES AS DIAGNOSTIC
AND THERAPEUTIC AGENTS
Justyna Bazan1,2, Kinga Gostomska3, Katarzyna Dudziak2
1
Wroclaw Medical University, Department of Medical Biochemistry 50-368 Wroclaw, ul. Chalubińskiego 10 ,
2
Bartla 5,
3
Wroclaw University of Environmental And Life Sciences, 50-375 Wroclaw, ul. Norwida 25,
Phage display is a powerful technique in medical and health biotechnology. This technology is
based on the fact that phage phenotype and genotype are physically linked. Phage display was
established by G. Smith in 1985as a method for presenting polypeptides on surface of lysogenic
filamentous bacteriophages. Nowadays this method is one of the most effective way for
producing large amounts of peptides, proteins, ligands, enzymes and antibodies.
By the use of this method the creation and fast and efficientsearchof antibody libraries is
possible. Phages and techniques based on this viruses found application in studying the proteinprotein or protein-ligand interactions, constructing of the antibody and antibody fragments and
improving the affinity of proteins to receptors.
The broad applicability of this technique allows its application in many areas of medicine
including the study of immunization process, vaccine design, investigation of allergen-antibody
interactions, cancer therapies and diagnostic.
Modified phages could be use for control of viral, fungal and bacterial infections such as HIV,
HBV, HCV, HSV-1, HPV, NSRV, Neisseria meningitidis, Staphylococcus aureus, Klebsiella
pneumoniae, Streptococcus pneumoniae, Mycoplasma pneumoniae, Pseudomonas aeruginosa
andCandida albicans.
References:
Bazan J, et al Hum Vaccin Immunother 2012; 8(12): 1817-1828
Clark JR et al. Expert Rev Vaccines 2004; 3:463-476
Mullen LM, et al. Trends Microbiol 2006; 14:141-147
Yang Q. et al. Vaccine 2005; 23:4088-4096
Acknowledgments:
This work is co-financed by the European Union as part of the European Social Fund.
51
VITAMIN D SIDE-CHAIN MODIFIED ANALOGS AND THEIR IMPACT
ON ACUTE MYELOID LEUKEMIA CELL LINES
Małgorzata Grudzień1
1
Department of Protein Biotechnology, Faculty of Biotechnology, Uniwersytet Wrocławski
Vitamin D3 is a compound which is synthesized in skin and transferred to the liver and kidneys
where is transformed to its biologically active form – 1,25(OH)2D3. Vitamin D plays a role in
maintaining calcium and phosphate homeostasis in vertebrate organisms and is a ligand for
vitamin D receptor (VDR). VDR after binding the ligand, heterodimerizes with retinoid X
receptor (RXR) which increases VDR stability and promotes nuclear import.
VDR belongs to the nuclear receptors superfamily and containsseveral conservative domains.
DNA-binding domain (DBD) is responsible for recognizing target sequences in genes. Ligandbinding domain (LBD) is a region which mediates heterodimerization.
Vitamin D and its synthetic analogs are tested as a potential therapeutics in leukemia treatment.
Vitamin D is capable to stop proliferation in acute myeloid leukemia (AML) and to start
differentiation of the cells, unfortunately concentrations of vitamin D which ensure those
activitiescause hypercalcemia and hyperphosphatemia. Many vitamin D analogs were
synthesized and tested on AML cell lines, several exhibit appropriate properties for becoming a
drug in cancer treatment.
Experiments with analogs PRI-1906 and PRI-1907 showed that these compounds display high
cell – differentiating activity. The aim of my future study is to test new, side-chain modified
analogs of vitamin D2 (PRI-5100, PRI-5101, PRI-5201and PRI-5202) and their influence on
HL60cell line differentiation and proliferation.
52
IN SEARCH OF THE PERFECT MATCH – COMPUTATIONAL ANALYSIS
OF POTENTIAL miRNA TARGET SITES
Adrian Grzemski1, Justyna Grabowska1
1
Poznań University of Life Sciences, Department of Genetics and Animal Breeding, Scientific Association of
Biotechnology Students OPERON
In 1993 Victor Ambros, Rosalind Lee and Rhonda Feinbaum discovered the role of microRNAs
in regulation of gene expression. These small RNA molecules, 20-24 nucleotides long, are
capable of reducing the amount of mRNA in cells by inducing their degradation, they inhibit
translation of peptides or even cause a decrease of peptide levels. Many efforts and resources
have been put into investigating the nature and mechanisms of such interactions. Scientists agree
that miRNA should be considered as a one of the most important gene regulation factors. It plays
a huge role in the development of obesity, oncogenesis and heart diseases.
Today, it is crucial to develop methods for quick and efficient searching for potential target sites
for whole set of miRNAs. In the past years many different algorithms that serve this purpose
were made. In this research we tested one of the most popular applications – miRanda. This
program evaluates each probable target place in terms of total complementary and calculates free
energy of optimal strand-strand interaction.
Using a set of experimentally proved interactions provided by miRTarBase, miRNA sequences
collected from miRBase and transcript 3'UTR sequences acquired with the UCSC Table Browser
data retrieval tool, we were able to verify sensitivity and efficiency of miRanda. We established
the optimal scanning parameters for receiving most reliable predictions of potential miRNA
target sites.
53
THE IMPORTANCE OF CELL HYDROPHOBICITY PSEUDOMONAS
AERUGINOSA IN BIOFILM FORMATION
Anna Guzy1, Grzegorz Czerwonka1, Wiesław Kaca1
1
Department of Microbiology, The Jan Kochanowski University, Kielce, Poland
Pseudomonas aeruginosa is the most common pathogen in patients with cystic fibrosis lung
infection (CF). Hydrophobicity of cell surfaces might be an important factor that have an
influence on adhesion to the host cells.Adhesion ability is one of the most significant features,
allowing bacteria to form biofilm.
The aim of this study was to determine the effect of cell surface hydrophobicity in biofilm
formation. Cell surfaces hydrophobicity was examined by BATH method (Bacterial Adherence
To Hydrocarbons) in 24 Pseudomonas aeruginosac linical strains. The ability of biofilm
formation was determined by crystal violet staining on microtiter plates.
Studies have shown that bacterial surface hydrophobicity may affect the ability to form biofilm.
Degree of adhesion Pseudomonas aeruginosa depends on microorganism isolate.
This analysis will allow to expand knowledge about Pseudomonas aeruginosapathogenesis in
respiratory system infections.Assay will determine degree of bacteria cell surface hydrophobicity
that might have an influence in biofilm formation observed in bacterial isolates from patients
with CF.
54
COMPARISON OF ANTICANCER ACTIVITY OF NATIVE AND BOILED
COELOMIC FLUID FROM DENDROBAENA VENETA
Sylwia Bilska1, Ewa Hordyjewska1, Beata Kliszcz1, Marta Fiołka2, Przemysław Kołodziej3
1
Scientific Students Group of Biotechnology “Mikron”,Faculty of Biology and Biotechnology, Maria CurieSkłodowska University, Akademicka 19, 20-033 Lublin, Poland
2
Department of Invertebrate Immunology, Faculty of Biology and Biotechnology, Maria Curie-Skłodowska
University, Akademicka 19, 20-033 Lublin, Poland
3
Department of Biology and Genetics, Faculty of Pharmacy, Medical University, Chodzki 4a, 20-093 Lublin, Poland
In an era of increased resistance to antibiotics new sources of natural therapeutic agents are still
sought e.g. in animals. Modern medicine often returns to the traditional solutions. From ancient
times earthworms are widely used as a remedy for variety of chronic diseases. The book Bencao
Gangmu wrote in 1593 describes 40 diseases which can be treated with components of
earthworms. What is important,biomass of these organisms is a rich source of proteins, peptides
and other active substances. In India traditional medicine earthworms are utilized in decoction
form against fever, stomach pain, neck pain, digestive and nervous disorders. Currently in China,
earthworm extract is used in treatment of over 80 diseases including hypertension, epilepsy,
cardiovascular disorders, as well as combating fever. Moreover, in Burma and Laos, bathing in
water containing coelomic fluid of earthworms is used to cure varicella. To expedite recovery
these organisms are roasted, powdered and eaten with coconut water. What is more, earthworms
decoctions are known as a source of vitality for women after childbirth. In Iran, earthworms are
roasted and added to bread to reduce and eliminate uroliths. Interestingly, earthworm ash in a
mixture of rose oil is used to stimulate hair growth, while the powder of earthworms is a
necessary component of many medicines for treatment bacterial and viral infections.
Our previous research has shown a significant activity of coelomic fluid (CF) from Dendrobaena
veneta against several human cancer cell lines, and it prompted us to preliminary define the
active compound character from CF.For this purpose native and post-immunization coelomic
fluid was boiled. High temperature caused decrease of CF activity. Obtained results may indicate
that proteins or their complexes denaturation occurred.
55
Innovative systems for in vitro cultures of animal cells
Hyra Katarzyna1
1
Politechnika Warszawska, Wydział Inżynierii Chemicznej i Procesowej, Zakład Biotechnologii i Inżynierii
Procesowej, ul. Waryńskiego 1, Warszawa. e-mail:katarzyna.hyra@gmail.com
Constant progress in the fields of biotechnology, bioengineering and biomedical sciences requires
continuous development of unconventional isolated animal in vitro cell and tissue cultures. There
is a need to obtain fully integral and functional tissues and cell units for tissue engineering and
regenerative medicine by developing innovative systems for cultures of this specific biological
material. The cultures of animal cells have recently allowed to produce many of the bioproducts
used in pharmacology as drugs or vaccines. This process enables to limit toxicological tests
conducted on animals by reducing studies of drug or physical factors effects on living organisms.
Appropriate culture conditions designed in this way allow to obtain tissue, that may be used in
regenerative medicine.
Innovative bioreactor systems, including single-use bioreactors, wave bioreactors and
liquid/liquid cultures, have been presented and discussed in the poster. Optimal in vitro culture
conditions according to physiology and morphology of animal cells, have been characterized. The
criteria for assessing the suitability of various cultures and potential ways of their development
have been described.
56
THE EXAMINATION OF FILAMENTOUS FUNGI ABILITY TO
SYNTHESIZE LIGNINOLYTIC ENZYMES AND THEIR POTENTIAL
CONTRIBUTION TO THE ELIMINATION OF TECHNICAL
NONYLPHENOL, 4-TERT-OCTYLPHENOL AND 4-CUMYLPHENOL
Tomasz Janicki1, Emilia Adamek1, Mariusz Krupiński1, Jerzy Długoński1
1
Department of Industrial Microbiology and Biotechnology, University of Lodz, Poland
Chemical compounds belonging to the group of EDCs have a negative impact on living
organisms by modulating their immune system. They get into the environment with waste water
and as a result of industrial processes. The ongoing development of the industry enhances this
negative process.Technical nonylphenol,4-tert-octylphenol and 4-cumylphenol
are
a compounds exhibiting structural similarity to natural hormones. This xenoestrogens are widely
used as surfactants, plasticizers and polycarbonate plastics.
The aim of the work was to study filamentous fungi ability to synthesize ligninolytic enzymes
and their potential contribution to the eliminationof technical nonylphenol, 4-tert-octylphenol and
4-cumylphenol. 40 strains of filamentous fungi from Museum of the Department of Industrial
Microbiology and Biotechnology, University of Lodz were the object of research.
In the first stage, the ability of fungal strains to oxidase ABTS, and thus to synthesize ligninases,
was assessed. Based on the results, 12 of the analyzed fungal strains were selected for further
experimental research concerning the enzymatic activity (laccase, lignin peroxidase and
manganese peroxidase). Six fungal strains demonstrated the ability to produce the analyzed
enzymes: four strains produced laccase, five – lignin peroxidase, one was able to synthesize
manganese peroxidase. In the next part of the study, the ability of the selected filamentous fungi
to grow in the presence of tested xenobiotics and their ability to eliminate xenoestrogens from the
growth medium were analyzed. In the last stage of the study, the enzymatic activity of the strains
in liquid cultures with the addition of tested compounds was examined.
57
POLYMIERIC NANOCAPSULES FOR CONTROLLED DRUG DELIVERY
Małgorzata Janik1, Joanna Odrobińska2
1,2
Wydział Chemii Uniwersytetu Jagiellońskiego;
The challenge for modern medical science is to develop advanced carriers for drug delivery,
which will enable the effective therapy with targeted release at a well defined place in the
organism. Among the materials polymeric structures such as liposomes and dendrimers play an
important role. There is also increasing importance of polymeric micro-and nanocapsules, that
allow for increasing of drug loading capacity, and change the encapsulated drug
pharmacokinetics during controlled release. It can also pursue for the minimize of the side effects
of treatment. Nowadays, the solid core capsules are replaced by capsules with liquid cores
stabilized with surfactants, what reduce their applications. That is why, usage of the amphiphilic
graft copolymers for stabilization the emulsion droplets (without surfactants) is one of the
solutions. There is a possibility of controlling molecular weight and architecture of the polymer
by using a controlled radical polymerization.
Synthesis of copolymers of different density and hydrophobic chain lengths enable to achieve
stable capsules with suitable sizes. This work includes considerations of the impact of grafting
density and length of the hydrophobic side chains of amphiphilic polyelectrolyte PAMPS-graftPVNp on capsules stability and size.
58
AN ANTARCTIC LIPASE LIPG7 – A NEW TOOL IN BIOCHEMICAL
ENGINEERING
Jarych Dariusz1,2, Florczak Tomasz1, Białkowska Aneta1
1
Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Łódź University of Technology,
Stefanowskiego 4/10, 90-924 Łódź, Poland,
2
Scientific Association of Biotechnology Students FERMENT
The Institute of Technical Biochemistry TULleads research on enzymes of organisms adapted to
operate at low temperatures. One of the most interesting enzymes is a lipase from mold
Geomyces sp. P7. This strain belongs to filamentous fungi and was isolated from soil therefore
lives at low temperatures and in conditions of relatively low water activity. Psychrophilic lipase
(isolated from this strain) due to its high catalytic activity at low temperatures and the unusual
specificity, offers new possibilities for the development of biotechnology. Wide range of
applications of these lipases include: as additives, in detergent and food production,
environmental remediation, and the production of drugs. The least mentioned application is of
particular interest as biocatalysis offers a stereo-specific and ecological methods alternative for
chemical processes.
Lipase lipG7 was found to have industrial potential as an
enantioselective biocatalyst as it is able to effectively
catalyse the enantioselective transesterification of
secondary alcohols. The LipG7 coding sequence has been
identified and cloned. The LipG7 protein has been just
heterologously expressed in Saccharomyces cerevisiae
BJ5465 and shown to exhibit the same characteristics as
the native protein.
Aims of this study was to transfer LipG7 gene to Pichia
pink system. Final goal was, optimization of isolation
procedure for pure preparation of lipase from Geomyces
sp. P7 (recombinant yeast of the genus Pichia pink).
Source: http://www.snapgene.com/resources/plasmid_files/yeast_plasmids/pPink-HC/ (access 30-10-2013)
References:
[1] Florczak T.; Daroch M.; Wilkinson MC.; Białkowska A.; Bates AD.; Turkiewicz
M.; Iwanejko LA.;“Purification, characterisation and expression in Saccharomyces cerevisiae of
LipG7 an enantioselective, cold- adapted lipase from the Antarctic filamentous
fungus Geomyces sp. P7 with unusual thermostability characteristics.” w: “Enzyme and
microbial technology”(2013) 53(1); ss. 18-24.
[2] Yingfeng A.; Jianfei J.; Wenfang W.; Anguo l.; Ribo H.; Yutuo W.; „A rapid and efficient
metod for multiple – site mutagenesis with a modified overlap extension PCR” w: „Applied
Microbiology and Biotechnology.”, (2005) 68: ss. 774-778
59
ANTIMICROBIAL PROPERTIES OF ZnO NANOPARTICLES
Marek Jotko1, Beata Kudawska2
1
Zachodniopomorski Uniwersytet Technologiczny w Szczecinie, Centrum Bioimmobilizacji i Innowacyjnych
Materiałów Opakowaniowych, ul. Klemensa Janickiego 35, 71-270, Szczecin, marek.jotko@zut.edu.pl
2
Materiałów Opakowaniowych, ul. Klemensa Janickiego 35, 71-270, Szczecin, beata.kudawska@zut.edu.pl
Nano-sized particles of ZnO have more pronounced antimicrobial activities than large particles,
since the small size and high surface-to-volume ratio of nanoparticles allow for better interaction
with bacteria. The main strategy of study is to use designed ZnO nanoparticles of different
surface morphologies and particle sizes. ZnO nanoparticles have a lower cost then other
antimicrobial agents and represent a class of material of increasing interest. Furthermore based on
the conclusions of the most recent risk assessment report of zinc oxide (2004) for zinc oxide
containing products, there is no concern for consumers for acute toxicity, skin, eye and
respiratory tract irritation, corrosivity and skin sensitisation. The design of ZnO particles are
well-known to affect the antimicrobial effect. Nanoparticles have selective toxicity to bacteria but
exhibit minimal effects on human cells.
The aim of this study was to determine the antimicrobial properties of ZnO particles.E.coli and
S.aureus were pre-grown on MacConkey agar (E.coli) and TSB containing 10% of sodium
chloride (S.aures) for 24h at 30°C. After incubation the biomass was suspended in sterile 0,85%
NaCl solution (0,5 McFarland scale). Then TSB medium was prepared (in the first falcon tube).
3ml of ZnO dispersion was added to 27g of broth. The next step was to prepare decimal dilutions
(0,1% and its dilutions from 10-1 to 10-9) of this dispersion in first falcon tube. The suspended
biomass was added to sterile flasks with broth TSB with ZnO (to all dilutions) in ration of 1:10
and stirred using magnetic stirrer for 15 minutes. After stirring, 100µl of each sample were plated
on mediums and incubated at 30°C for 24h. Cell concentration was expressed as colony-forming
units (CFU) per ml and determined by making serial decimal dilutions and plating on
MacConkey agar (E.coli) and TSB (S.aureus). Results are presented as average of three samples
with standard deviation.
60
INNOVATIVE ADDITIVES IN PAPER PRODUCTION
– UNIQUE PROPERTIES
Marek Jotko1, Beata Kudawska2
1
Materiałów Opakowaniowych, ul. Klemensa Janickiego 35, 71-270, Szczecin, marek.jotko@zut.edu.pl
2
Materiałów Opakowaniowych, ul. Klemensa Janickiego 35, 71-270, Szczecin, beata.kudawska@zut.edu.pl
The environmentalimpact of world industry production, is currently significant.Modern
technologies ofpaper production has led to highconsumption of planet wood resources andlarge
amountsof waste producedevery year.This cause the global difficulties in securing adequate
quantities of raw materials to customers from paper industry, as well as the utilization of the
increasing amount of waste paper. However, increasedenvironmental awarenessof peopleon the
planetforcesincreasedgovernment regulationof waste management andsustainabledevelopment of
thepulp and paper industry.
Paper production in the world has increased more than seven times over the past 60 years, has
finally reached the level of 360 million tons in 2006 and its still rising about 1.4% every year.
Because of this facts it seems necessary to look for other products that may replace wood
cellulose.Biorefining is seen as a process closely related to the energy industry but, it has a much
broader meaning. It also used to describe any method of purification and refining of natural
substances that cause them to obtain the relevant properties. Currently agro-food byproducts such
as sugar beet pulp or corn straw are used mainly as animal feed or combustible material.
However the high content of cellulose allows to consider other more profitable applications of
this biobased byproducts. The solution assumes "full" use of byproducts and their evaluation
through ecofriendly biorefining via appropriate enzymatic digestion after the thermo process of
distending and fiberizing of starting material. That allow the isolation of valuable cellulose fibers
which can be use as a wholesome substitute of typical wood fiber pulp in production of paper and
paperboard with a improved mechanical strength parameters and the stability of these traits in
varying conditions of temperature and humidity. Mixtures of different low-cost natural products
with different properties will lead to obtaining product with unique functionality properties while
maintaining similar threshold price for the finished product which is currently produced on the
basis of waste paper. After the biorefining process non-cellulosic components will remain
possibly unchanged that will allow their direct, or after purification and processing, application in
other industries.
61
GENERATION OF TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR
NUCLEASES (TALENs) TARGETING Adam10GENE
Szymon Juszkiewicz1, Maria Czarnek1, Jarosław Jucha1, Witold Nowak2, Szymon Czauderna2,
Joanna Bereta1
1
Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University
in Kraków, Kraków, Poland
2
University in Kraków, Kraków, Poland
Transcription activator-like effector nucleases (TALENs) are widely used as a highly efficient
targeted genome engineering tool. TALENs are composed of programmable, sequence-specific
DNA binding moduleslinked to a DNA cleavage domain of a type II restriction endonuclease FokI.
Such constructs should induce double-strand breaks followed by non-homologous end-joining
(NHEJ) or homologous recombination (HR) at specific genome sites. One of the possible
applications of TALENs is making knockout animals or cell lines by targeted gene inactivation [1].
We applied TALEN technology to knock out gene encoding ADAM10 (a disintegrin and
metalloprotease 10), one of the major sheddases. More than 40 ADAM10 substrates have been
identified [2] including proteins involved in cell growth and differentiation (Notch, EGF, ErbB2)
as well as those responsible for the immune system functioning. ADAM10 has been suggested to
promote development of cancer as well as inflammatory diseases and therefore, is considered as a
potential drug target [3]. ADAM10-silencing by siRNA is a method of choice for studying its role
in the cell. However, siRNA-mediated silencing has multiple disadvantages such as non-complete
and unstable level of silencing and possible non-specific immunostimulation of cells [4].
ADAM10-deficient cell lines are thus desirable models for research on the role of ADAM10 in
cancer progression and metastasis, whileTALEN technology emerges as a promising alternative
for siRNA silencing.
The engineering of TALENs involves few steps: designing the specific DNA binding domains,
obtaining and assembling of proper modules, and testing the activity of final TALENs. Here we
present our modified protocol for generating functional TALENs disrupting Adam10 gene.
References:
[1] A. J. Bogdanove and D. F. Voytas, “TAL Effectors: Customizable Proteins for DNA Targeting,”
Science, vol. 333, no. 6051, pp. 1843–1846, Sep. 2011.
[2] K. Endres and F. Fahrenholz, “Regulation of alpha-secretase ADAM10 expression and activity,”
Exp. Brain Res., vol. 217, no. 3–4, pp. 343–352, Oct. 2011.
[3] H. C. Crawford, P. J. Dempsey, G. Brown, L. Adam, and M. L. Moss, “ADAM10 as a therapeutic
target for cancer and inflammation,” Curr. Pharm. Des., vol. 15, no. 20, pp. 2288–2299, 2009.
[4] C. Shen, A. K. Buck, X. Liu, M. Winkler, and S. N. Reske, “Gene silencing by adenovirusdelivered siRNA,” Febs Lett., vol. 539, no. 1–3, pp. 111–114, Mar. 2003.
62
Viruses- a weapon against a cancer
Martyna Kabacińska1, Kamila Karoń1, Przemysław Olejnik1
1
Koło Naukowe Studentów Biotechnologii „OPERON”, Uniwersytet Przyrodniczy w Poznaniu
Low efficiency of the conventional mathods In treating cancer disease and their negative impact
on the human body are couses of searching for alternative ways of therapy. Reserches relating to
the use of viruses in cancer therapy started in 1950s, but the concept was born ealier – in 1912.
The wild type viruses was used for all of these treatment regimens and gaves as a result a number
of complications, therefore in the 70‟s and 80‟s dropped the oncoviruses therapy, but in the 90‟s
the genetic engineering has brought a breakthrough in researches.
Oncolityc viruses infect and lysis cancer cells and also doesn‟t interfere with normal cells. They
can become an alternative for previous methods of treatment, thank to their selectivity and
specificity. We can notice three functions of this viruses: increase sensitivity of cancer cells to the
chemotherapeutics, induce specific anit-tumor immunity and may be carriers of the factors(e.g.
proteins and prodrugs) involved in death of the cancer cell
New treatment for use oncolityc viruses brings hope to hundreds of thousands of patients with
cancer.
63
DERMATHOPHYTHES – ENZYMATIC VIRULENCE FACTORS
Katarzyna Kadzewicz1
1
Student‟s Society of Biotechnology Gdansk University of Technology
So far, scientist have described almost 100 000 fungus, but only about 40 000 has pathogenic
feature. One of the most common are Dermathophytes. It is pathogenic, keratinophilic group of
fungi, which the main feature is their capacity to invide keratinized tissue (hair, nails, skin) of
humans or animals. The results of colonization of the karotinized layers is dermatomycoses - one
of the most common infection. According to place where they exist we can classified it into three
anamorphic genera Epidermophyton, Microsporum, Trichophyton.
In recent year, the interesting about secreted enzymes by fungi is increasing. We know that the
enzymes are very important virulence factors, but we don‟t have enough knowledge about
substrate specificity and inhibitors. The dermathophythes product enzymes like proteases and
lipases, which are help it to penetrate hosts issues, by the changing the structure of cell
membrane,
Proteases refers to a group of enzymes whose catalytic function is to hydrolyze (breakdown)
proteins into peptides or free amino acids. They are also called proteolytic enzymes or
proteinases. Classification of proteases basis on their mode of action and their active sites
Other important enzymes which we can classified as a virulence factor are phospholipases. A
group of enzyme whose hydrolyzes phospholipids into fatty acids and other lipophilic
substances.They play an important role by hydrolyzing glycero host cell membranes, but also are
likely to be responsible for maintaining the normal function of the cell membrane of fungi.
64
THE IMPORTANCE OF SURFACE HYDROPHOBICITY OF PROTEUS
MIRABILIS CELLS IN THEIR ABILITY TO FORM BIOFILM ON SOLID
SURFACE
Klaudia Kałuża1, Grzegorz Czerwonka1, Wiesław Kaca1
1
Department of Microbiology, The Jan Kochanowski University, Kielce, Poland
Proteus mirabilis is an important etiological factor of the urinary tract infections (UTIs), where in
12%
of
cases
the
infections
are
with
complications.
This
bacterium
has an important virulence factors for colonization in the specific environment, including
the ability to cells adhesion, which are considered to be one of the characteristics of
the formation of biofilms.
The purpose of the research is to evaluate the impact of surface hydrophobicity
of the twelve clinical and laboratory strains Proteus mirabilis in their ability to grow
in biofilm. To this end, the ability to create the biofilms by method of microtitration plates and
evaluation of cells surface hydrophobicityby method's BATH (Bacterial Adherence to
Hydrocarbons)was specified.
Research has shown that hydrophobicity at the surface of the bacteria affects
their capacity to grow in biofilm. On the basis of assays it is assumedthat the degree of adhesion
of bacteria Proteus mirabilis is a feature depending on the strain of microorganism which is
characterized by a variety of growth conditions. Different adhesion properties are correlated
witha variable part of the structure of a O-antigen of lipopolysaccharide, whose chemical
structure is a feature depending on the clinical strain. The differences in hydrophobicity in
bacterial cells, which affect the amount of formed biofilm, were observed as well.
Study complements knowledge about pathogenesis of the bacteria Proteus mirabilis
in urinary tract infections. Furthermore, the thesis introduced the phenomenon of adsorption and
presented to what extent the cells surface hydrophobicity of these bacteria influence on the
phenomenon of biofilm formation which may be observed in the majority long term catheterized
patients.
65
EVALUATION OF ANTIBIOTIC SUSCEPTIBILITY OF
UROPATHOGENIC ESCHERICHIA COLI STRAINS
Ewelina Kamińska1, Monika Wawszczak1, Wioletta Adamus-Białek2
1
Biotechnology Academic Circle – Mikroby; Department of Microbiology, Jan Kochanowski University, Kielce,
Poland
2
Department of Enviroment Protection and Modelling, Jan Kochanowski University, Kielce, Poland
Rapid growth of antibiotic resistance of pathogenic bacterial species is alarming. Uropathogenic
Escherichia coli strains (UPEC) belong to this group. This issue is very important because of
cosmopolitan nature of this species. Most strains of Escherichia coli are harmless, they are part of
normal human gut flora, but some of them are dangerous and can cause gastroenteritis and
urinary tract infections (UTI). These bacteria are widespread in environment and possess the
mobile genetic elements, which takes part in spreading of virulence factors. A rise of bacterial
resistance to antibiotics complicates treatment of urinary infections.
Aims of research:

Analysis of antibiotic susceptibility of UPEC strains to 16 antibiotics most commonly
used in UTI.

Identification of strains producing extended-spectrum beta-lactamases (ESBL)

Identification of strains with unique patterns of antibiotic resistance

Comparison of antibiotic resistance patterns of studied strains to current data.
Materials and methods:

Collection of 127 Escherichia coli strains, isolated urine samples of patients suffering
from urinary tract infection, from the wards of Military Teaching Hospital No. 2, Medical
University of Lodz, Poland, in the years 2006 to 2008.

16 antibiotics from different classes: Penicillins, Cephalosporins, Carbapenem,
Aminoglycosides, Quinolones, Nitrofurans, Sulfonamides, derivative of folic acid.

Determination of antibiotic susceptibility testing by disk diffusion method and Double
Disc Synergy Test (DDT )
Expected results:
Identification of antibiotic susceptibility patterns of studied E. coli strains to antibiotics used in
urinary tract infection treatment will be performed. We also expect identify the strains producing
extended-spectrum beta-lactamases (ESBL). The results will help identify high-risk strains and
the directions of spread of drug resistance in the population of UPEC.
66
IN VITRO EVALUATION OF CYTOTOXIC EFFECT OF
POLYELECTROLYTE NANOPARTICLES ANT THEIR APPLICATION
AS A DRUG DELIVERY SYSTEM
Alicja Karabasz*1, Monika Bzowska1, Joanna Bereta1, Krzysztof Szczepanowicz2
1. Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian
University, Gronostajowa 7, 30-387 Cracow, Poland
2. Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, 30-239 Cracow,
Poland
Nanotechnology concerns the production and application of engineered particles or system which
have at least one dimension less then 100 nm in length. There are many potential applications of
nanomaterials in biomedicine but in our studies we have focused on nanoparticles as a drug
delivery system.
Use of conventional chemiotherapeutic agents is limited because of their poor water solubility,
short lifetime in circulation, and toxic side effects on normal cell. Nanoparticles are regarded as
an effective candidate to overcome this limitations. Nanocapsules have been designed to improve
the biodistribution and solubility of cancer drugs thereby anticancer agents are less toxic for
normal tissue and also intracellular concentration of drugs can be increased. Moreover
nanomaterial surface is susceptible to chemical modifications that can improve their circulation
time in the bloodstream and is useful in active targeting of encapsulated drug to the tumor.
In vitro analysis of nanoparticles toxicity and their efficacy as anti-cancer drug delivery system is
essential step in preclinical studies of nanomaterials. The main aims of our studies are: 1) to
evaluate the cytotoxic effect of nanocapsulesas a drug delivery system in vitro 2) to verify anticancer activity of encapsulated anticancer agent – paclitaxel. In this research we are using
paclitaxel-incorporated nanocapsules which are based on a liquid core withoppositely charged
polyelectrolyte multilayer prepared by the technique of sequential adsorption of polyelectrolytes
called Layer-by-Layer technique.
To determinate cytotoxic effect of nanoparticles and anti-cancer activity of encapsulated
paclitaxel on viability of 4T1 cell (mouse breast cancer) MTT assay and measurement of LDH
activity were performed. We also studied cell morphology by light microscopy and verified
apoptosis mediated cell death by DNAladdering assay. This analysis is supporting on cells
treated with nanoparticles without paclitaxel, paclitaxel-incorporated nanocapsules or free
paclitaxel .
67
COLLAGEN – SOURCES AND THE PRODUCTION METHODS
Kamila Karoń, Jakub Jagielski, Wojciech Piechowiak
The aim of our poster is presentation of the most important, recent researches in the field of
collagen. We want to show you various methods used to extract the matter and describe very
interested source, fish wastes. There are many ways of receiving collagen from the tissues.
We have selected three main examples: salt precipitation, acid isolation and enzyme isolation.
Each of them contains different stages and reagents. It is worth to say why we have choose this
topic. Therefore, recently collagen has gained on popularity because of diversified application
and impact on many areas of human life and production.
Subsequently we would rather you made interested in potential opportunities of using the
substance in the future. Nowadays the substance finds its own level in several areas of life,
mainly medicine and cosmetics.
68
STEVIA REBAUDIANA – SWEETNESS IN NATURE
Kinga Karp
Faculty of Biotechnology and Food Sciences, Łodz University of Technology, Łódź ul. Wólczańska 171/173, 90-924
Łódź, Poland Scientific Association of Biotechnology „Ferment‟
Stevia rebaudiana is a plant of Asteraceae family, native to South America (Brazil and Paraguay).
Commonly is known as sweet leaf or simply stevia, is widely grown for its sweet leaves. As a
sweetener and sugar substitute, stevia's taste has a slower onset and longer duration than that of
sugar.
The plant hasa very high content of sweeteners and therefore is used to sweeten beverages and
foods. It is characterized by a very low calorific value compared to a traditional sugar. The
research conducted on 12 patients with type 2 diabetes mellitus have shown beneficial stevia‟s
activity effects on lowering blood glucose.
In addition, an important effect of consumption stevioside which is contained in leaves of stevia
is also its ability to reduce blood pressure in patients with hypertension.
69
RECOMBINANT BIOPHARMACEUTICALS – WHERE DO THEY COME
FROM?
Dominika Helena Kępska
Institute of Technical Biochemistry Faculty of Biotechnology and Food Sciences Łódź University of Technology,
Wólczańska 171/173, 90-924 Łódź, Poland, Scientific Association of Biotechnology Students FERMENT
According to classical definition biopharmaceuticals are approved for human use chemical
compounds, mostly glycoproteins. Presently, they exemplify the fastest developing sector in the
field of pharmaceutical industry and represent a number of protein classes like growth factors,
hormones, antisera, enzymes, enzyme inhibitors and monoclonal antibodies.
Obtaining of biopharmaceuticals requires appropriate expression systems, which are needed for
protein expression and it‟s posttranslational modifications. Among these modifications the most
essential is glycosylation. In mammalian cells, the oligosaccharides present in glycoproteins are
linked to a protein with O-or N-glycosidic bond. Type of N-glycosylation is started by movement
of a preassembled oligosaccharide to an asparagine residue present in unfolded or partially folded
polypeptides. While O-glycosylation includes the transfer of a single sugar residue to a serine or
threonine hydroxyl group of a fully folded protein. Composition and heterogeneityof glycans on
the surface of therapeutical proteins may affect their: yield, bioactivity, stability against
proteolysis, immunogenicity, solubility and clearance rate from circulation.
Nowadays, prevailing production of protein drugs is conducted in mammalian platforms, due to
their natural ability for expression of human-like glycosylation. However, during the last ten
years, a plant-based expression platform for the generation of recombinant glycoproteins with
defined N-glycans was developed. Plant-based expression systems have several advantages such
as safety, the easiness of scale-up and low cost for industrial production.
Recombinant biopharmaceuticals are applied in many different therapeutic approaches for
unfulfilled medical needs. Frist produced biopharmaceuticals were recombinant human insulin
and human interferon. Now, therapeutic glycoproteinsare crucial for treatment of diverse cancer
types, autoimmune diseases and replacement therapies- as enzyme and hormone substitutes. They
also take part in protection from infectious diseases.
70
RNA STRUCTURE OF THE INFLUENZA A VIRUS- CHEMICAL
PROBING
Julita Kęsy
Poznan University of Medical Sciences
Influenza A virus belongs to Orthomyxoviridae family viruses and is a causative agent of
epidemic and occasionally pandemic flu. Its genome is segmented and consists of eight
single-stranded negative-sense RNA molecules folded into separate rod-shaped ribonucleoprotein
complexes (RNPs). The focus on the structure of the virus arises from its important role in the
infection cycle. There are many techniques that have found application in RNA mapping and
allow to investigate genomic organization under a variety of conditions. Chemical probing is one
of the most popular methods. Chemical reagent used to modify RNA may be a small organic
molecule, metal ion or an enzyme. The experiment yields a wide range of information on basestacking, hydrogen bonding, electrostatic environment in the vicinity of the particular base,
solvent accessibility to the backbone, local nucleotide flexibility and dynamics. Rational
combination of these techniques gives ability to interrogate every single base in the strand and
predict higher-order structure formation. The aim of the research is to gain a better understanding
of the structural basis of the RNP functioning and to utilize it in potential anti-viral drug
development.
71
MOLECULAR MECHANISM OF GDF-15 AND SDF-1- INDUCED
ANGIOGENESIS – NON-CANONICAL ROLE NRF2 TRANSCRIPTION
FACTOR
A.Grochot-Przęczek, D. Klóska, A. Józkowicz, J. Dulak
University
Blood vessel formation is a tightly regulated process, crucial for organism development and
normal tissue repair. It also plays a role in physiological events in adults, for instance in
oogenesis, menstrual cycle and hair growth. Blood vessel formation contributes to many
disorders, wherevexcessive (cancer, rheumatoid arthritis) or impaired vascularization (coronary
artery and peripheral arterial diseases) causes disease development or treatment failure. SDF-1
and GDF-15 are the molecules potently inducing angiogenesis.SDF-1 is a CXC chemokine,
initially cloned from bone marrow stromal cells.9 It exerts numerous activities and is widely
expressed in the body. SDF-1 exhibits angiogenic activity through its potent effect on endothelial
and progenitor cell migration and differentiation. It is released at site of the injury and plays a
major role in recruitment of progenitor cells to ischemic tissues. GDF-15, also referred to as
macrophage inhibitory cytokine-1 (MIC-1), is a member of transforming growth factor-β (TGFβ) superfamily, playing multifunctional roles in numerous physiological and pathological
processes. Its membrane receptor is not known yet. GDF-15 is a potent survival and antiapoptotic factor. The molecular mechanism of GDF-15-induced angiogenesis is not fully
elucidated and remains further investigations, however, it was proposed, that GDF-15 may inhibit
p53 signaling, thereby activating hypoxia inducible factor 1α (HIF1α) pathway, which stimulates
potently angiogenic response.
Preliminary data shows that angiogenesis induced both by SDF-1 and by GDF-15 is driven only
in presence of Nrf2 transcription factor. Nrf2 is a master regulator mediating adaptive response to
cellular stress. It was also demonstrated by siRNA knockdown of Nrf2 and overexpression of
Keap1 that disruption of Nrf2 signaling impairs angiogenic response of endothelial cells driven
by VEGF and insulin-like growth factor-1 (IGF-1).
The aim of present project was to investigate the molecular mechanism of proangiogenic activity
of growth differentiation factor 15 (GDF-15) and stromal cell-derived factor-1 (SDF-1) and to
demonstrate that angiogenesis driven by these factors depends on Nrf2 function not related to its
transcriptional activity.
Experiments were done on GDF-15 and SDF-1-stimulated human aortic endothelial cells
(HAECs) with siRNA knockdown expression of Nrf2.
Conclusion: Lack of Nrf2 transcription factor impairs particular stages of blood vessel formation
(migration, conversion into tip cell, sprout elongation, lumen formation) driven by GDF-15 and
SDF-1.
72
INFLUENCE OF CANDIDA ALBICANS METABOLITES ON ACTIN
REARRANGEMENT IN HEp2 CELLS
Paulina Knobloch1
1
Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University,
Cracow, Poland
Candida albicans is a commensal fungal organism which can be found among
the majority of human population. However, under some circumstances, like immunodeficiency,
it can overgrow and attack the host. The attack is supported by many adaptive abilities of C.
albicans, among others by morphological transition to hyphal forms, expression of particular cell
surface proteins or the secretion of enzymes with hydrolytic activity.
The influence of C. albicans and C. albicans metabolites on HEp2 human larynx epithelial cells
was investigated by a research group lead by E. Segal from the Sackler School of Medicine, TelAviv University, Tel-Aviv, in cooperation with the Bruce Rappaport Faculty of Medicine,
Technion, Haifa.
In vitro studies showed remarkable changes in the cellular morphology and actin cytoskeleton
organization in HEp2 cells as the result of their interaction with C. albicans as well as with the
fungal metabolites. Afterward the active subfraction of C. albicans secreted factors was
characterized and the effect on HEp2 cells of combined treatment with the fungal metabolites and
bisindolylmaleimide (BIM) or cytochalasin D (CyD) was investigated.
73
TET PROTEIN (TEN-ELEVEN TRANSLOCATION PROTEINS) – NEWLY
DISCOVERED REGULATORS OF EXPRESSION AND THEIR ROLE IN
CANCEROGENESIS
Anna Kołodziejczak1 , Katarzyna Tomela1, mgr inż. Bartosz Frycz1, prof. dr hab. Paweł
Jagodziński1
1
Department of Biochemistry and Molecular Biology, Poznań University of Medical Sciences
For decades, the epigenetics and its impact on cancer development has taken an important place
among the problems of modern science. Although the mechanisms of one of the most common
epigenetic modifications such as DNA methylation seem to be fairly well understood, it is the
reverse process - the DNA demethylation that is still a mistery. A newly discovered protein family
TET (TET1, TET2, TET3) may be a breakthrough revelation in this matter [1].
Owing tothe „zinc finger” domain CXXC located at the N-terminal, TET protein readily
associate with CpG islands near the site where transcription begins. After anchoring they may
catalyze reaction of metylocytosine hydroxylation(5mC) to hydroxymetylocytosine (5hmC).
Then inactive methylated DNA becomes transcriptionally activeagain. It has recently been
proved, that TET1 is also involved in the recruitment of repressor protein such as PRC2 which
inhibit expression of genes associated with cell differentiation. On the basis of these experiments
it has been found that TET proteins play a major role, e.g. in maintaining the stem cells and
neurogenesis [2].
The significance of TET proteins for the proper development of cells may be confirmed by the
fact that for the first time they were discovered in the form of a fusion gene with MLL in a patient
with leukemia. It had earlier been known that the growth of many types of cancer is encouraged
by the changes of methylation profile related to global hypomethylation and hypermethylation of
CpG islands in the promoter regions of tumor suppressor genes. In solid tumors, i.a. gliomas,
colorectal cancer, breast cancer and melanoma, decreased levels of 5hmC correlates with a
decrease in TETprotein expression [3]. This phenomenon also occurs in the case of gastric
cancer, and studies carried outin our department indicate that there is a significant difference in
the level of TET1 protein expression between healthy tissue and cancer tissue in patients
suffering from this type of cancer, which we are currently trying to correlate with
histopathological data.
REFERENCES
[1]„Tet family proteins and 5-hydroxymethylcytosine in development and disease”, Li Tan and Yujiang Geno Shi,
Development 139, 1895-1902 (2012)
[2]“Dual functions of Tet1 in transcriptional regulation in mouse embryonic stem cells”, Hao Wu, Ana C. D‟Alessio,
Shinsuke Ito, Kai Xia, Zhibin Wang, Kairong Cui, Keji Zhao, Yi Eve Sun, and Yi Zhang, Nature 2011 May 19;
473(7347): 389–393
[3]“TET Family Proteins and Their Role in Stem Cell Differentiation and Transformation”, Luisa Cimmino, Omar
Abdel-Wahab, Ross L. Levine and Iannis Aifantis, Cell Stem Cell 9, September 2, 2011
74
THE ANALYSIS OF CHANGES IN THE CONTENT AND ITS
DERIVATIVES ANALYSIS IN CUCUMBER (CUCUMIS SATIVUS L.)
SEEDS ONE YEAR AFTER OSMOCONDITIONING
Marta Rakowiec1, Izabela Kołodziejczyk1, Rafał Szewczyk2, Malgorzata M. Posmyk1
1
Department of Ecophysiology and Plant Development, Faculty of Biology and Environmental Protection, University
of Lodz ul. Banacha, 12/16, 90-237 Lodz martab@biol.uni.lodz.pl
2
Department of Industrial Microbiology and Biotechnology, Faculty of Biology and Environmental Protection,
University of Lodz ul. Banacha, 12/16, 90-237 Lodz
Our previous experimentshave shown that exogenous melatonin (MEL) applied into
dicotyledonous seeds could be a good biostimulator.In the case of cucumber seeds MEL applied
at the optimal concentration of 50 μM, increased their germination in suboptimal temperature 15oC and extend the range of germination temperatures up to 10oC. Moreover, the seedlings
grown from the seeds osmoprimed with MEL were much more vigorous especially under
environmental stress condition (chilling, heavy metal pollution).
In the presented work the efficiency of MEL application methods into the seeds was verified.
Osmopriming (PEG -2 MPa at 25oC for 4 days) was chosen as optimal for cucumber. Four
variants of seeds were compared: control – non-treated ones, conditioned with water and
conditioned witch MEL water solution of 50 and 500 μM (cucumber seeds: C, O, OMel50,
OMel500).
The changes in the content of MEL and the appearance of its potential metabolites were
monitored by HPLC-MS quantitative and qualitative analyses of seed extracts for one year
starting from the moment of conditioning and idoleamine application.
The control cucumber seeds and these conditioned with water contained a small amount of MEL:
7-40 ng/gFW. However, it is worth mentioning that in the seeds not treated with exogenous MEL
(variants: C, O) seasonal changes in this indoleamine concentration were observed – a significant
increase in the winter months.
The osmoconditioning techniques supplemented with MEL proved to be a highly effective tool to
force this idoleamine into cucumber seeds. MEL levels increased proportionally to its applied
concentration of MEL. It was noticed MEL was metabolized in the seeds storedfor 1 year at room
temperature in darkness. Three of its derivatives were determined: hydroxymelatonin, 3-(2hydroxyethanal)-5-metoxyindol, and cyclic hydroxymelatonin.
Research work financed in the years 2011/2014 by the NCN N N310 111940
75
INFLUENCE OF PLATINUM NANOPARTICLES ON HUMAN PRIMARY
KERATINOCYTES
Konieczny Piotr1, Goralczyk Anna1, Szmyd Radoslaw1, Skalniak Lukasz1, Koziel Joanna1, Pyza
Elzbieta1, Filon Francesca Larese2, Crosera Matteo2, Cierniak Agnieszka1, Zuba-Surma Ewa1,
Borowczyk Julia1, Laczna Eliza1 Drukala Justyna1, Klein Andrzej1, Jura Jolanta1
1
From Jagiellonian University, Krakow, Poland
2
From University of Trieste, Italy
The platinum group elements (PGEs) represent a new kind of environmental pollutants and a new
hazard for human health. Since their introduction as vehicle exhaust catalysts, their emissions
into the environment have considerably grown compared with their low natural concentration in
the earth crust. PGEs emissions from vehicle catalysts are in the form of nanometer sized
particles (PtNPs). These elements, both in their metallic form or as ions solubilized in biological
media, are now recognized as potent allergens and sensitizers. Human skin is always exposed to
toxic particles. As there is no so far data concerning the impact of PtNPs on primary
keratinocytes in the present study, we addressed the question whether polyvinylpyrrolidonecoated (PVP) PtNPs may have any negative effects on this type of cells.
In the study two sizes platinium nanoparticles were used: 5.8 nm and 57 nm in concentration
6.25, 12.5 and 25 µg/ml. Both types of nanoparticles were protected with polyvinyl-pyrrolidone
(PVP). Primary keratinocytes were treated for 24 and 48h then cytotoxicity, genotoxicity,
morphology and metabolic activity were investigated. Moreover, we analyzed antimicrobial
properties of PtNPs against Gram-positive and Gram-negative bacteria.
76
LOWERED CHOLESTEROL LEVEL OR DECREASED CX43
EXPRESSION INHIBITS TGF-β1-INDUCED SMAD SIGNALING IN
HUMAN BRONCHIAL FIBROBLASTS FROM ASTHMATIC PATIENTS
Milena Kosińska*1, Izabela Borek1, Damian Ryszawy1, Katarzyna Anna Wójcik1,2, Jarosław
Czyż1, Marta Michalik1
1
Jagiellonian University, Faculty of Biophysics, Biochemistry and Biotechnology, Department of Cell Biology,
Gronostajowa 7, 30-378 Kraków, Poland
2
Jagiellonian University Medical School, Department of Medicine, Skawińska 8, 30-031 Kraków, Poland
Smad signaling pathway plays a crucial role in TGFβ1-induced fibroblast-to-myofibroblast
transition [FMT], a process involved in airway wall remodeling during bronchial asthma [1].
Activation of Smad proteins in human bronchial fibroblasts [HBFs] participates in this process
and up-regulates the expression of α-SMA protein - a marker of myofibroblasts [2]. In search of
extrinsic and intrinsic modulators of FMT, we investigated the effects of zaragozic acid A,
lovastatin and connexin 43 [Cx43] function on Smad pathway activity in asthmatic HBFs
stimulated by TGF-β1.
Lovastatin inhibited TGFβ1-induced nuclear translocation of p-Smad2, which correlated with a
decrease in α-SMA expression and the fraction of myofibroblasts in TGFβ1-treated HBFs
populations. A similar effect was observed when TGF-β1 - treated HBFs were concomitantly
subjected to siRNA silencing of Cx43 gene. In contrast, 18-α-glycyrrhetinic acid - a non-specific
inhibitor of gap junctional coupling, insignificantly decreased nuclear translocation p-Smad2, but
considerably reduced the fraction of myofibroblasts in HBFs cultures.
TGFβ1-induced Smad-dependent signaling in asthmatic HBFs remains under the control of
cholesterol-dependent pathways and gap junctional channel-independent Cx43 function. Gap
junctional coupling may participate in FMT of asthmatic HBFs in a Smad2-independent manner.
References:
[1] Hinz B. Formation and function of the myofibroblast during tissue repair. J Invest Dermatol.
127. 526–537. 2007
[2] Evans et.al. TGF-beta1-mediated fibroblast-myofibroblast terminal differentiation-the role of
Smad proteins. Exp Cell Res. 282(2). 90-100. 2003.
77
SURVIVAL RATE OF MICROORGANISMS ISOLATED FROM STAFF
ROOMS OF THE FOOD PRODUCTION PLANT IN PRESENCE OF
ESSENTIAL OILS
Magdalena Koszałkowska1, Teresa Krzyśko-Łupicka2
1
scholarship holder of the project „Stypendia doktoranckie - inwestycja w kadre naukowa województwa opolskiego”
co-financed by the European Union under ESF. University of Opole, mkoszal@o2.pl;
2
Department of Biotechnology and Molecular Biology, University of Opole, Kard. Kominka Str. 6a, 45-035 Opole,
teresak@uni.opole.pl
The monitoring of the environment related to food production spaces is needed to get safe and
good quality products. The monitoring of microbiological purity of the environment around the
technological spaces is indicated of both the ISO 22000 and the code of Good Manufacturing
Practice (GMP). Microbial contamination in staff rooms of food production plants could be taken
with the employees into the production space. It is therefore essential to clean and disinfect not
only the production areas. Recently an increased importance of natural disinfectants such as
essential oils was observed.
The aim of this study was to estimate the survival rate of microorganisms isolated from staff
rooms in food production plant in presence of thyme and rose essential oils.
Materials were biomass of microorganisms isolated from staff rooms as ladies‟ cloak room (CL),
men‟s cloak room (CM), technical storeroom (ST) and canteen (CA) collected by swabs method.
Swabs were collected from 4 walls. The results were averaged and given in CFU/m2. The
influence of rose and thyme essential oils in 1 and 2 % concentrations on microbiological
survival was evaluated in comparison to negative control (without disinfectants) and peracetic
acid (in concentration of 0,5 %) as positive control. The survival rate was tested by the classic
plate method and results was given as the percentage.
In canteen and cloak rooms dominated bacteria (87-100%). Technical storeroom was the only
place where a high level of fungi (98% the total microbial population) was noticed. Rose oil in
1% concentration and thyme oil in 2% concentration inhibited 100 % of microorganisms from
canteen (CA) and both cloak rooms (CM, CL) and its activity was comparable to peracetic acid.
Less antimicrobial activity of ecological substances was observed for technical storeroom (ST),
where rose and thyme oils in all tested concentrations stimulated the growth of microorganisms
(relative to negative control).
78
SOS RESPONSE REPRESSOR OF PARACOCCUS AMINOPHILUS MAY
INFLUENCE TRANSCRIPTION OF ANTISENSE REGULATORY RNA
Łukasz Kowalski, Jakub Czarnecki, Dariusz Bartosik
Department of Bacterial Genetics, Institute of Microbiology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw,
Poland
Bacterial genomes consist mainly of protein-coding genes, however recent transcriptome studies
performed in various bacterial species indicate the presence of extensive antisense transcription
in these organisms. As proven, many antisense transcripts have physiological significance and
they constitute and important level of gene expression. cis-encoded regulatory RNAs bind
complementary target mRNAs and may influence their stability (positively or negatively) or
interfere translation (e.g. by masking of RBS sequence).
We have performed preliminary analyses of the SOS regulon of bacterium Paracoccus
aminophilus JCM 7686 (Alphaproteobacteria), which is a methylotrophic strain with a
multipartite genome. SOS regulon is a global regulatory network, highly conserved in bacteria.
Under normal conditions, transcription of genes of the regulon is repressed by LexA protein that
binds specific DNA sequence motif (SOS-box), situated within the SOS genes promoters. When
DNA damage occurs RecA protein supports autocatalytic cleavage of LexA, leading to release of
transcription of the SOS genes involved in DNA repair.
In silico analysis of the P. aminophilus genomic sequences revealed that the putative SOS-boxes
are situated not only within the promoter regions, but also, in a few cases, (i) within open reading
frames and (ii) downstream of the regulated genes. The latter case strongly suggests possibility of
post-transcriptional regulation of gene expression. A good example of such phenomenon seems to
be a ligA-recG operon, containing two downstream placed SOS-boxes (genetic organization of
this locus is conserved in all paracocci). We have purified LexA protein of P. aminophilus and we
have shown binding of the represor to both predicted SOS-boxes. In silico analysis of the
nucleotide sequence revealed that the LexA binding sites overlap a -30 box of a promoter, driving
transcription towards the end of the recG gene. We propose that this inducible promoter may be
responsible for transcription of a cis-acting regulatory RNA, with a potential to influence stability
of ligA-recG transcript under SOS response.
79
INFLUENCE OF ALTERNATIVE DISINFECTANTS ON THE SURVIVAL
RATE OF MICROORGANISMS ISOLATED FROM TECHNOLOGICAL
AREAS IN FOOD INDUSTRY
Łukasz Kręcidło1, Teresa Krzyśko-Łupicka2
1
PhD student of Biology and graduated student of Biotechnology in Agriculture and Food Production, University of
Opole, mag18-89@o2.pl;
2
Department of Biotechnology and Molecular Biology, University of Opole, Kard. Kominka Str. 6a, 45-035 Opole,
teresak@uni.opole.pl
The clean environment of food production without any pathogenic and spoilage microorganisms
is necessary to preserve a good quality of products. Disinfection is one of the most important
processes in technology of food production, which allows to control hazard of contamination.
Many of food production plants use chloride disinfectants but these substances have a negative
impact on the environment.
The aim of this study was assessment of the influence of alternative disinfection substances on
survival rate of microorganisms isolated from production surfaces in a food industry.
The research material was the biomass of microorganisms isolated from production areas in the
food production plant by swabs method. The samples were taken from 4 walls from production
hall (HP1), technological surfaces (HP2) in the main production hall and aseptic production halls
(AC and AU). The results were given in CFU/m2. The influence of alternative disinfectants as
rose and thyme oils (in concentration of 1 and 2 %) on survival rate of microorganisms was
evaluated in relative to negative control (without disinfectants) and positive control (peracetic
acid in concentration of 0,5 %). The survival rate was tested by the classic plate method. The
results were given as the percentage.
Rose oil in 1% concentration was effective as peracetic acid and totally inhibited (100%) biomass
from aseptic areas (AC, AU). In production hall (HP1, HP2) the most effective was a thyme oil in
1 % concentration (97-100% inhibition). In the laboratory tests, rose and thyme oils (in 1%
concentration) inhibited the growth of microorganisms, but only in pilot-scale studies it will be
possible to estimate their effectiveness.
80
TITANIUM DIOXIDE-COATED PAPER – ANTIMICROBIAL EFFICACY
Beata Kudawska1, Marek Jotko1
1
Center of Bioimmoblization and Innovative Packaging Materials, West Pomeranian University of Technology in
Szczecin, ul. Klemensa Janickiego 35, 71-270 Szczecin, Poland
The photocatalytic properties of titanium dioxide (TiO2) are well known and have many
applications in the industry. In recent years, the sterilization withTiO2has attracted attention as an
alternative to thedisinfection of surfaces, air and water.The activity of TiO2 has been shown to be
capable of killing a wide range of Gram-positive and Gram-negative bacteria, fungi, protozoa,
algae, mammalian viruses and bacteriophage. The antibacterial activity of TiO2 has been found to
be due to production of strong oxidizing power when illuminated with UV light.. On exposure to
ultraviolet irradiation, TiO2 releases reactive oxygen species (hydrogen peroxide and hydroxyl
radicals) which cause degradation of the cell wall and cytoplasmic membrane of microorganism.
Degradation leads to leakage of cellular contents, cell lysis and then complete mineralisation of
the organism.
The aim of this study was to create on the paper an antimicrobial layer containing titanium
dioxide and cationic starch. Coatings were applied to the reverse side of the paper using a
sprayer, which provides a wet film thickness of 3µm and a dry grammage of 1g/m2. The wet
sheets were transferred to an oven, dried at 70°C for 4 minutes and then exposed to UV light. The
paper was tested then on two model microbes, Gram-positive bacteria Staphylococcus aureus and
Gram-negative bacteria Escherichia coli. To test the antimicrobial efficacy of the paper, the
square samples (1.5 cm × 1.5 cm) were cut from both the untreated and TiO2 treated papers. The
samples were placed in culture medium and incubated for a 2h at 37 °C.
After incubation Minimal Inhibitory Concentration (MIC) was used to calculate the growth
inhibition.
81
BACTERIAL CHROMIDS – ESSENTIAL EXTRACHROMOSOMAL
GENETIC ELEMENTS
Katarzyna Kuźmicz, Jakub Czarnecki, Dariusz Bartosik
Poland
Research in the field of genomics has produced data suggesting that the structure of many
bacterialgenomes is more complex than was previously assumed. Many bacteria were found to
bearadditional, large, autonomic replicons, which (like chromosomes) carry a pool of housekeeping (HK) genes. Unlike typical plasmids, such replicons are necessary for the viability of
their hosts,and for this reason they were initially defined as "secondary chromosomes". However,
this name isinadequate since these replicons possess many characteristics typical of plasmids:
they containplasmid-like replication systems (REP) and other genetic modules of plasmid origin.
Bioinformaticanalyses of these replicon sequences indicated that they were generated by the
transfer of geneticinformation from chromosomes to plasmids co-residing in the cell. Due to their
dualistic properties,they were reclassified into a separate, newly distinguished group with
properties of bothchromosomes and plasmids: the chromids.
Chromids share a number of major characteristics: (i) considerable size (they are usually
thesecond largest replicons in the cell, being larger than the chromosomes of the some bacteria),
(ii)the presence of plasmid-type replication systems, (iii) G+C nucleotide contents comparable
withthat of chromosomes, (iv) codon usage in coding sequences similar to that of chromosomes
(pointsiii and iv indicate long-term coevolution of chromosomes and chromids), (v) the presence
of HKgenes, typical of chromosomes (removal of chromids from the bacterial cell causes a lethal
effect),and (vi) the presence of adaptive genes (typical for plasmids), enabling adaptation of
bacteria to agiven ecological niche.
The prevalence of chromids in bacteria and their conserved character within certaintaxonomic
groups suggests an important role for these replicons in the evolution of bacteria. Theformation
of multi-replicon genomes, in which the basic genomic information is split betweendifferent
replicons, is probably beneficial to bacteria. For example, the presence of an additionalreplication
system, enabling more rapid duplication of the genetic information, should permitmaintenance of
a larger genome while keeping a high rate of cell division. This hypothesis issupported by the fact
that the average size of chromid-bearing bacterial genomes (5.73 Mb) exceedsthat of genomes
that have a single chromosome (3.38 Mb). Moreover, it has been shown thatthe frequency of
dimer generation by bacterial chromosomes increases exponentially in relation totheir size;
therefore, the reduction of chromosome size allows minimization of this phenomenon. Chromids
are characterized by their high variability (resulting mainly from the low density ofhousekeeping
genes), and are therefore considered to be specific "training sites" in which differentevolutionary
variants are "tested", which leads to structural variability of multireplicongenomes in different
strains of the same genus.
82
DEPENDENCY BETWEEN GENE p53 AND NEOPLASIA
Wojciech Langwiński1, Kacper Nowacki1
1
Uniwersytet Przyrodniczy w Poznaniu
Gene p53 is one of the most examined genes which protein product is widely regarded as a tumor
suppressor. It has only been detected in multicellular organisms. In a human p53 is located on the
short arm of chromosome 17th, has 20.3 kpz. of length, and its protein product consists of 393
aminoacids.
Mutations, in DNA sequence encoding p53 suppressor, are the most frequent reasons for all kinds
of cancer development.
The natural (wild) protein is a transcription factor which, by activating gene p21, stops the cell
cycle until the DNA strand is repaired. The second mechanism is based on the cell suicideapoptosis. Both of these ways lead to the inhibition of cell multiplication.
Due to damages, an inappropriate protein accumulates in the body and does not work as a
transcription factor, which results in the enlargement of the number of tumor cells. The reestablishment of natural function of p53 protein could be the significant response for increase of
the cancer cases number.
83
STRUCTURE AND PROPERTIES OFLIGNOCELLULOSIC COMPLEX
Mariusz Lesiecki1, Przemysław Piotr Olejnik2, Adrian Czerniak1
1
Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences;
2
ScientificAssociation of Biotechnology Students "Operon",Poznań University of Life Sciences.
The lignocellulose complex are main compound of plant dry matter, so it's known as
lignocellulose biomass. The lignocellulose is the most abundant raw material on the Earth. It is
composed of celluloses, hemicelluloses and lignin. Particularly celluloses are consider as great
substrate to produce biofuels.
Cellulose is great source of glucose, but not available to the most of microorganism. Some of
them can produce group of enzymes, cellulases, which are able to convert celluloses from
lignocelluloses biomass to glucose. In industry this step limit whole production of some process
and generate costs, e.g. bioethanol production. The knowledge of structure and specific character
of lignocellulose biomass allows for matching property processes and enzymes to develop
optimum conditions for hydrolysis. It also help us using this material as substrate to
biotechnology processes
84
LIGHT IT UP
Michał Lewandowski, Katarzyna Kubiak
Lodz University of Technology, Faculty of Biotechnology and Food Science, Institute of Technical Biochemistry, 90924 Stefanowskiego 4/10 Lodz, Scientific Association of Biotechnology Students “Ferment”
Nanocelullose is a very useful biopolymer. Because of its unique properties we use it in
medicine, for example, as a wound dressing and implants. In Nature bacteria producing NC are
living on decanting fruits and use this biopolymer as a protective shield against harmful
environmental conditions, like UV radiation. The aim of our project is to find out how light
influences the growth of bacterial nanocellulose produced by Glucoacetobacter xylinus. We
prepared fifteen flasks and dived them in groups of three. Each group was placed in various
conditions. The two of them were control groups- they have grown in a box, without the access of
daylight. The third one was exposed to electric light twenty-four hours per day. The forth one was
experienced day-night light changes as it was placed on the windowsill. The last group was
exposed it to short UV light fluxes every day.
85
YEAST BIOSENSOR OF TNT
Michał Lorentowicz1
1
After series of tragic terrorist assassinations which was done at the beginning of 21 st century,
scientists have started to think how to protect the population against terrorists. One of the most
interesting ideas in the field of biotechnology was to involve popular yeast Saccharomyces
cerevisiae. Team of scientist from Philadelphia‟s Temple University created yeast biosensor
which detects DNT (2,4-dinitrotoluene). DNT is a waste material in TNT (2,4,6-trinitrotoluene)
production, which is present in every explosives containing TNT. This compound can be detected
by some of mammals through the sense of smell. Yeast biosensor was obtained by insertion of a
gene coding for rat‟s olfactory receptor protein into pheromones receptor protein‟s gene in
Saccharomyces cerevisiae cells. To visualize binding between odour molecule and receptor a
gene of fluorescent protein was inserted, in such a way to be expressed after receptor‟s activation.
In effect, activated yeast cell shines in a green colour. Screening of transformed cells with TNT
enables a selection of a cells which could be a component of a TNT biosensor.
86
BIOTECHNOLOGICAL METHODS OF REDUCING THE INFLUENCE
OF ABIOTIC STRESS FACTORS ON PLANTS
Monika Olszówka1, Kamil Maciąg2
1
olszowka.monika@gmail.com, Scientific Group of Biotechnology KUL, The Faculty of Biotechnology and
Environmental Sciences, John Paul II Catholic University of Lublin, al.Krasnicka 102, 20-718 Lublin, Poland
2
maciagkamil@gmail.com, Scientific Students Group of Biotechnology “Mikron”, The Faculty of Biology and
Biotechnology, Marie Curie-Sklodowska University, ul. Akademicka 19, 20-033 Lublin, Poland
The plants are able to grow in different environmental conditions, but the molecular explanation
why some plants successfully grow in e.g. high salinity or near polar regions is less obvious.
Responsibile for that capability are genes that code special metabolic pathways or sometimes one
small mutation. Unfortunately, the majority of humans desired to obtain plants able to grow in
wide environmental conditions spectrum. The big group of researchers works to understand how
plants can grow in unfriendly conditions such as: drought, high salinity, low or high temperature,
toxic concentration of metals and ions, gases (SO2, NOX, O3) and other stress factors. Almost all
of the resistance ability is a consequence of a presence of the special genes.
Nowadays, agricultural statistics data show growing trend to increases crops area. Still observed
phenomenon of soil erosion forces humans to understand resistance mechanisms and improved
new features of the crops. That is necessary to produce enough amount of food for cost-effective
prices. As the result, biotechnological methods of plants improvement are more popular and more
frequently used in practice.
One of the evermore popular methods of transfer of stress-induced genes is by genetic
engineering methods e.g. a gene encoding a hybrid-proline-rich protein from the pigeon pea
transferred to Arabidopsis and adaptive to tolerate a multiple abiotic stress; a DREB/CBF factor
from wheat, which is responsible for tolerance to drought and cold stress, TSRF1- an ERF
transcription factor from tomato that improves tolerance to drought in rice and a sugarcane
ethylene responsive factor (ERF), which additionally enhances salt and drought tolerance in
tobacco plants.
There is only few examples of successful discoveries in this area. The increase of genetic
modification popularity and amount of funds causes the increase of novel and spectacular
discoveries.
87
THE ARRANGEMENT OF LUTEIN AND ZEAXANTHIN IN POPC LIPID
BILAYER
Krzysztof Makuch1
1
Jagiellonian University, Department of Computational Biophysics and Bioinformatics
Lutein and zeaxanthin are two most abundant carotenoids in eye and plant lipid membranes.
These carotenoids can perform many different functions such as free radical scavenging,
structural functions in protein folding or light absorption in energy antennas.
The aim of these studies is verification of lutein and zeaxanthin arrangement within the POPC
model lipid bilayer using molecular dynamics method. Previously conducted studies for the lutein
indicated mixed - that is perpendicular or parallel to the surface of the membrane - orientation of
the lutein. Because of the small number of repetitions and low frequency of entrance of the lutein
into the bilayer, it was necessary to repeat the simulation for large number of systems.
Constructed systems differ in the initial arrangement of the carotenoids in relation to the surface
of the membrane. Results show that both lutein and zeaxanthin seem to be able to take
perpendicular and parallel orientations. However orientation of lutein and zeaxanthin are
stabilized by different interactions with POPC molecules.
Another approach to determine arrangement of carotenoids in lipid bilayer involves inserting
lutein and zeaxanthin directly into the membrane. Earlier laboratory research showed that both
lutein and zeaxanthin form aggregates in liposomes. Such aggregates due to the lack of mobility
cannot reorient as freely as monomeric forms. Moreover change of absorption spectra lets them
to act as energy filters for different wavelengths. In our studies we observed formation and even
entrance of aggregates into the POPC model bilayer.
All systems were simulated for 100ns with GROMACS molecular dynamics package, using
OPLS-AA forcefield. This research was supported in part by PL-Grid Infrastructure.
88
THE USE OF BACTERIOPHAGES AGAINST LISTERIA
MONOCYTOGENES IN DAIRY INDUSTRY
Jamruszka Tomasz1, Mazurkiewicz Natalia1
1
Scientific Association of Biotechnology Students „Operon”, Poznan University of Life Sciences
Listeria monocytogenes is widely distributed in the environment and, during the last decade, was
recognized as an important food-borne pathogen. Various foods, such as meat, milk and other
dairy products, and vegetables contaminated with L. monocytogenes have been linked with
human listeriosis. Listeriosis occurs primarily in certain high-risk groups, including pregnant
women, neonates, and immunocompromised adults.
Lactic acid bacteria play an important role in the manufacturing of fermented foods, especially
dairy products. These bacteria are responsible not only for the development of flavor and texture
but also for the preservation of many products. Unfortunately, a big obstacle in the production of
dairy products are the bacteria Listeria monocytogenes.
The aim of latest important study was to introduce the endolysin-encoding genes from Listeria
bacteriophages into lactococcal starter organisms in order to obtain organisms with
biopreservation properties against L. monocytogenes.
It turned out also that an effective way to preserve dairy products from undersirable Listeria
monocytogenes is an application of bacteriophages for specific killing of the bacteria. It seems to
be a big approach to enhance food safety.
89
MicroRNA IN ASTHMA
Beata Narożna1
1
Poznan University of Medical Sciences, Laboratory of Molecular and Cell Biology, Poznan, Poland.
Asthma is common chronic inflammatory disease of the airways caused by bronchial
hyperresponsiveness. It is estimated that more than 300 millions of people worldwide have been
diagnosed with asthma, over 250 thousand people die every year. Asthma is a complex disease,
determined by both genetic and environmental factors.
MicroRNA (miRNA) are small non-coding RNA molecules, 18 to 25 nucleotides long,
responsible for regulation of protein translation through the mechanism called RNA interference.
They‟re mostly responsible for development, differentiation, function and apoptosis of cells.
MicroRNA are also involved in the inflammation process.
Existing research suggests that miR-126, miR-146 and miR-133 may partake in pathogenesis of
asthma, mostly through regulation of processes such as lung remodeling, inflammation (by
increasing cytokines levels), excessive mucus production, bronchial hyperresponsiveness and
hypertrophy of smooth muscles. Using a molecule called antagomir, which blocks specific
miRNA and results in slower development or inhibition of asthma symptoms, creates a new
therapeutic solutions for this disease.
Research of miRNA in asthma enables accurate determination of asthma pathogenesis
mechanism and creates new diagnostic and therapeutic options.
90
PRODUCTION OF PROPIONIC ACID ON DIFFERENT SOURCES OF
CARBON BY PROPIONIBACTERIUM SPP.
Monika Kowalska1, Natalia Mazurkiewicz1, Joanna Pawlicka2, Katarzyna Czaczyk2
1
2
Scientific Association of Biotechnology Students „Operon”, Poznan University of Life Sciences
Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences
Propionic acid bacterium (Propionibacterium spp.) is a Gram-positive pleomorphic rods, which
are characterized in living in the relative anaerobic conditions and not producing spores. Their
characteristic feature is the ability for the production of metabolites, such
as propionic acid, acetic acid, carbon dioxide, vitamin B12. Classic propionic acid bacteria
are naturally present in milk, dairy products such as Swiss-type cheeses, bread sourdoughs
or silages.
In the study was used a strain of Propionibacterium freudenreichii ssp. shermanii 1, belonging to
the collection of its own Department of Biotechnology and Food Microbiology, Poznan
University of Life Sciences in Poznan.
Two seven-day cultures in duplicate were grown on casein medium with different carbon sources.
The source of first culture was glucose, second glycerol. Aim of this study was to examine the
profile of metabolites produced by Propionibacterium spp. on media with different carbon
sources, but also to search the amount of propionic acid prodused by this bacteria.
The growing interest in the production of chemical compounds by biotechnology tends to find
cheaper sources of carbon. Glycerol is a by-product in the production of biodiesel. The research
and literature data confirm the possibility of using it for the production of propionic acid. Applied
appropriate source of carbon has a very large impact on the course of fermentation – shapes, the
dynamics of production and profile of metabolites in the post-culture fluids, and thus affect the
economics of the process.
91
WHAT IS IN A MICROSCOPE IMAGE? CONVOLUTION AND
DECONVOLUTION
Sylwia Nicpoń
1
1
Scientific Students Group of Biochemistry, Faculty of Biology and Biotechnology, Maria Curie-Skłodowska
University, Akademicka 19, 20-033 Lublin
Confocal microscopy currently enjoys great popularity in the research community. Images
obtained by using confocal microscopy are characterized by an unprecedented resolution,
contrast, sensitivity in the detection of light, and the ability to obtain a series of cross-sections of
the object. The advantage of laser scanning confocal microscope is also a possibility to create
three-dimensional visualization and reconstruction. These featuresallowto obtain images of better
quality and to perform more comprehensive data analysis. The digital images can be further
improved by means of image processing which can reduce noise, and, at least to some extent,
enhance contrast and resolution. The image of an object obtained by a confocal microscope is
distorted by the point-spread function of the instrument. In order to restore the actual image, a
deconvolution algorithm can be used.
In my study, I used the artificial digital image stack, which was subjected to convolutionwith a
point-spread function typical for the confocal microscope.Subsequently I performed the
deconvolution using Huygens (SVI, http://www.svi.nl/)or AutoQuant (Media Cybernetics,
http://www.mediacy.com) software. Here I report a comparisonof the results obtained using the
two programs in terms of accuracy and image quality.
References:
[1] Korczyński J., New dimension in microscopy – laser scanning confocal microscope,
Kosmos, 2013, 62, 2,ss.149-160
[2] Wolny A., Visualizations in the world of light microscopy, Kosmos, 2013, 62, 2,
ss. 161-170
[3] Schoonderwoert V., and et al, Huygens STED Deconvolution Increases Signal-to-Noise
and Image Resolution towards 22nm, Microscopy Today, 2013, 21, 6, ss.38-44
92
PHOSPHOLIPIDS MODIFICATION IN PAECILOMYCES MARQUANDII
UNDER ALACHLORE EXPOSURE.
Justyna Nykiel1,2, Adrian Soboń1, Mirosława Słaba1, Przemysław Bernat1, Jerzy Długoński1
1
Department of Industrial Microbiology and Biotechnology, University of Lodz, Banacha 12/16 90-231 Lodz,
Poland.
2
Biotechnology and Microbiology Student‟s Society “Bio-Mik”.
Alachlor [2-chloro-N-2,6-diethylphenyl-N-(methoxymethyl)acetamide]is a one of the most
frequency applied pre-emergence herbicide. Despite its documented harmful effects on humans
and animals, it is still widely used in the world including Europe, Japan and the USA. Alachlor
like others chloroacetamides can inhibit synthesis of lipids and fatty acids and also interrupts cell
metabolism.Little is knownabout the effectsof alachlor on microorganisms capable to degrading
this xenobiotics, in particular microorganismslipidome, that plays a important role in maintaining
cell membrane.
In this workwe studiedtheeffectof alachlor on phospholipids of P.marquandii strain, isolated from
high metal polluted environment (Silesia, Poland).
Liquid cultures of P.marquandiiwith the addition ofalachlor were incubated on a rotary shaker at
28oC for 5 days. The washed fresh mycelia were homogenized and extracted with an organic
solvent mixture of chloroform and methanol (2:1, v/v). Next, all samples were prepared for
analysis of phospholipidsby HPLCMS/MS.
A total of 29 lipid species inP. marquandii mycelium was identified. It has been shown that tested
herbicide cause modification in the profile of phospholipids, involving changes in the percentage
of main classes (phosphatidylcholine and phosphatidylethanolamine).
Lipid profile changes,observed withexposure toalachlor, relied ona rise in the levelof
phosphatidylcholineand an increase inthe overallsaturationcan be attributed todefense response of
fungal cells against alachlor toxic action.
This study was supported by the Grant of the National Centre for Science in Cracow, Poland, No
UMO-2011/01/B/NZ9/02898.
93
USEFUL TOXINS
Łukasz Olejnik
Institute of Technical Biochemistry Faculty of Environmental Biotechnology Łódź University of Technology,
Stefanowskiego 4/10, 90-924 Łódź, Poland,Scientific Association of Biotechnology Students FERMENT
Batrachotoxins (BTX)belongs to the group of neurotoxicsteroidalalkaloids. The name of this
toxin comes from the Greek word "batrachos" (βάτραχος) which means frog. BTX was
discovered in 1960s in skin extracts from a Colombian poison-dart frog (Phyllobates terribilis).
This toxin consist mainly of a steroid skeleton and oxazapane ring. The scientists whoseparated
the potent toxic alkaloids fraction from batrachotoxin and determined chemical properties were
Bernhard Witkop and John Daly. For a long time considered that BTX cannot be synthesized in a
laboratory. It has changed when in 1991 batrachotoxin A was synthesized by Michio Kurosu,
Lawrence R. Marcin, Timothy J. Grinsteiner, and Yoshito Kishi.
Batrachotoxin is oneof the most dangerouspoisonsin the world. 1-2 µg/kgof this toxinis alethal
dosefor an adult human. Batrachotoksynacausesmuscle paralysis, impairment of respirationand
cardiac arrest.To this dayit isused bySouth AmericanIndiansto poison thetipsof arrows
andblowers. The poisoncan be effectivelyfor a period ofabout one year.
But the poison that can kill, it can also be used as a medicine. It has been proved that the proteins
contained in the venom of the frog is able to prevent cancer, blocking the virus mutations and
even have an analgesic effect. Anotherpotenttoxinistetrodotoxin from the puffer fish (Takifugu).It
seemsthat the effect of tetrodotoxinoffsets theeffectsof batrachotoxin.
94
APLICATION OF PECTIN IN FOOD INDUSTRY
Przemysław Piotr Olejnik1, Mariusz Lesiecki2, Adrian Czerniak2
1
ScientificAssociation of Biotechnology Students "Operon",Poznań University of Life Sciences;
2
Department of Biotechnology and Food Microbiology , Poznań University of Life Sciences
Pectins are natural components of plant biomass. Generallypectinsform a complex with cellulose
which is known as protopectin, and forms the binder of the cell walls . Particularly large amounts
of pectins are present in fruits , such as blackberry, gooseberry , and citrus and apples.
Pectinsare obtained from food industry wastes, such as pomace from apples and other fruits.
These preparations are used as food and drugs additives.
Pectin is a plant-derived substance is the best gelling agent for jams and fruit jellies . As a natural
component of fruits, its addition to food products, help to retain the organoleptic characteristics
of final product.
95
MLPA- METHOD OF IDENTIFICATION OFGENE MUTATIONS
Bartłomiej Olszewski
1
Lodz University of Technology, Faculty of Biotechnology and Food Sciences,ul. Wólczańska 171/173, 90-924 Łódź,
Introduciton: Multiplex Ligation-dependent Probe Amplification (MLPA®) is a method which
detects aberrant copy numbers of up to 45 specific nucleic acid sequences in one simple PCR
reaction, using a single PCR primer pair.Applications include the detection of exon
deletions/duplications in e.g. the human BRCA1, MSH2 and MLH1 genes; detection of trisomies
such as Down syndrome; characterisation of chromosomal aberrations in cell lines and tumour
samples; SNP and mutation detection; DNA methylation analysis and relative quantification of
mRNA.
Objective: Use MLPA method in identification SNP mutation in human genom.
In this method are used hybridizing specific probes for complementary tested sections of DNA.
This probes are constructed from two fragments, that after hybridizing to tested section are
ligated and then used as a matrix for DNA polymerase.
Conclusion: MLPA is primarily a method which identifies deletions/duplications - it is not a
suitable method to detect unknown point mutations. Nevertheless, MLPA is able to discriminate
known point mutations, as probes can be designed so that the ligation site of a probe is located
directly at the site of the point mutation. Ligation will then only occur if the target sequence is the
perfect reverse complement of the probe sequence, resulting in a decreased fluorescent signal in
the case of a mismatched DNA sequence. Probes can be designed either to detect the wild type
sequence or the SNP/point mutation in question.
96
BIODEGRADATION OF BISPHENOLA A BY FILAMENTOUS FUNGI
EMERICELLA CORRUGATA AND MUCOR SP.
Milena A. Piątek1, Jerzy Długoński2
1
Biotechnology, University of Lodz, Poland
2
Endocrine Disrupting Compounds (EDCs) are exogenous agents that interfere with action,
binding, elimination, transport, synthesis and secretion of hormones in the living organism by
mimicking natural hormones. One of the most harmful and dangerous EDCs is bisphenol A. BPA
2,2-bis(p-4-hydroxyphenyl)propane is used for the production of epoxy resins, phenol resins,
polyacrylates and polycarbonates. The addition of BPA to food packaging and plastic materials
such as bottles, pacifiers, and toys for children poses a real risk and causes leakage of the
compound to surface water. This xenobiotics is resistant to degradation so finding
microorganisms capable of BPA elimination from the environment could be a good solution.
The objective of this study was to identify the degradation products of BPA by strains Mucor sp.
IM 6324 and Emericella corrugata IM4659.
The culture of filamentous fungi Emericellacorrugata IM4659 derived fromNigeria and Mucor
sp. IM6324 isolated from municipal waste in Moscow was conducted in Sabouraud medium.The
analysis was done by ultra-liquid chromatography coupled with tandem mass spectrometry
(UPLC MS/MS) in a negative mode based on BPA. The initial concentration of bisphenol a in the
growth medium was 50 mg/l.
After 5-day of incubation fungal mycelium was homogenized, then the xenobiotic and
degradation products were extracted. The tested strains: IM4659 and IM6324 showed an ability
to degrade BPA almost completely. The main metabolites obtained in the fungal culture were 4hydroxybenzoic acid, 4-hydroxybenzaldehyde and 4-hydroxyacetophenone. It has allowed typing
monocyclic products derivedfrom the decomposition ofBPA.
Strains IM4659 and IM6324 showed an ability to degrade bisphenol A. The presented study
confirmed a split in the methyl bridge in the structure of BPA.
This study was supported by the grant of the National Centre for Science in Cracow, Poland, no
UMO-2011/01/B/NZ9/02898.
97
SCREENING FOR MICROORGANISMS CAPABPE OF BISPHENOL A
DEGRADATION
Milena A. Piątek1, Adrian Soboń1, Jerzy Długoński2
1
Biotechnology, University of Lodz, Poland
2
Bisphenol A or 2,2-bis(p-4-hydroxyphenyl)propane (BPA) is an organic compound composed of
two phenol rings connected by a methyl bridge with two methyl functional groups.BPA was
synthesized for the first time in the „90s and it was originally used for hormone replacement
therapy as a synthetic estrogen. Currently, it is used in the production of plastics, polyesters, such
as polycarbonates and synthetic resins, as well as an antioxidant in cosmetics and food.Plastics
Polycarbonate (PC) is characterized by high strength and ductility, which is why they are used for
the production of digital media, electrical and electronic equipment, and the manufacture of food
packaging and bottles. A controversial aspect is use of BPA to produce high quality transparent
baby bottles or pacifiers, as small amounts of BPA may be released from theepoxy resins or
polycarbonate resins into foods and beverages.
The aim ofthis study was todetermine theability of selectedfilamentous fungito eliminateBPA.
The cultureof individualfilamentous fungiwas conducted in WHI3 or Sabouraud medium,
depending on the growth requirements. The initial concentration of bisphenol a in the growth
medium was 50 mg/l. After 168 h incubation the samples were extracted using ethyl acetate and
analyzed by liquid chromatography coupled with tandem mass spectrometry (LC MS/MS).
In order to find a microscopicfilamentous funguscapable ofbisphenolAdegradation, 20strainswere
tested. Fungalstrainsisolated frompollutedenvironmentsmost effectivelyeliminatedbisphenol
Afrom the culture medium. The strains: IM 6324, IM 6449, IM 6471 and IM 6482 showed an
ability to degrade BPA almost completely.
It has been shownthat thefilamentous fungifrom differentenvironmentsare capable
ofefficientelimination ofbisphenolA.The conductedresearchallowedselectingstrains which
efficientlyremoveBPAfrom the culture medium.
This study was supported by the grant of the National Centre for Science in Cracow, Poland, no
UMO-2011/01/B/NZ9/02898.
98
IDENTIFICATION OFA LIFESTYLE-DETERMINING CHROMID OF
PARACOCCUS AMINOPHILUS JCM 7686 (ALPHAPROTEOBACTERIA)
Jakub Czarnecki, Emilia Prochwicz, Łukasz Dziewit, Dariusz Bartosik
Poland
Chromids constitute a newly distinguished group of bacterial extrachromosomal replicons. They
are essential for their hosts, in contrast to plasmids, which are dispensable (under most
conditions) and can be easily removed from bacterial cells. Numerous studies on chromids have
indicated that these replicons carry genus-specific genes which allow to survive their host strains
in the natural habitat. The presence of chromids correlates with high metabolism plasticity of
bacterial strains and capability to occupy unique ecological niches.
Paracoccus aminophilus JCM 7686 is a methylotrophic strain isolated from soil polluted with
N,N-dimethylformamide. Its multireplicon genome is composed of a chromosome (3.6 Mbp) and
eight extrachromosomal replicons (pAMI1-pAMI8), ranging in size from 5 kb to 438 kb. We
have conducted comprehensive bioinformatic and functional analyses to determine which of
these replicons meet the definition of chromid.
Our results revealed that pAMI5 (294 kb) of P. aminophilus JCM 7686 is a typical chromid
carrying a set of housekeeping genes, therefore it could not be removed by incompatibility from
the JCM 7686 cells. On the contrary, pAMI6 (206 kb) could be readily removed, however strains
lacking this replicon grew poorly on LB medium and lost the ability to grow on minimal media.
It seems obvious that pAMI6 is essential for the strain survival in the natural habitat, therefore we
suggest that this replicon should also be regarded as chromid.
To support this hypothesis we have performed additional analyses of pAMI6. The obtained
results revealed that pAMI6 contains genetic information, which significantly shapes
methylotrophic pathways of the JCM 7686 strain. By mutational analysis we have identified
within pAMI6 two equivalent copies of tmm gene, encoding trimethyloamine monooxygenase –
the first enzyme enabling conversion of trimethyloamine to methyloamine by monooxygenases
pathway. We also found that pAMI6 encodes glutamate-mediated pathway for methylamine
oxidation (product of the process is formaldehyde) and tetrahydrofolate-dependent formaldehyde
oxidation pathway.
In silico predictions concerning utilizationof methylated amines were confirmed by LC-MS/MS
analysis, which revealed that mainly pAMI6-encoded genes are induced under growth of P.
aminophilus on trimethyloamine. These findings confirmed the crucial role of pAMI6 in
providing adaptive and niche-specific genetic information to its host.
99
INVESTIGATING THE CROSS LINK BETWEEN TORC2, LIPID
METABOLISM AND DNA DAMAGE RESPONSE
Sara Przetocka1,3, Ghadeer Shubassi2, Mery Piña2, Marco Foiani2
1
Faculty of Biochemistry, Biophysics and Biotechnology, Department of Microbiology, Jagiellonian University,
Krakow
2
FIRC Institute of Molecular Oncology Foundation (IFOM), Milan
3
The Scientific Club of Biotechnology Students “Mygen”
One hallmark of cancer and gaining cells is an unstable genome, which results from DNA
damage. Following genotoxic stress, the evolutionary conserved DNA damage response (DDR) is
activated. In buddying yeast Saccharomyces cerevisiae the DDR is mediated by two PI3 kinases:
Mec1 and Tel1. This mediation of DDR results in activation of signal transduction pathways,
which involves the Rad53 and Chk1 kinases. It recently has been reviled that DDR can be
regulated not only from the nucleus but also by the processes taking place in cytoplasm like
autophagy or osmotic stress. Both processes at certain stages are downregulated by TOR2
complex and possibly involve actin filament dynamics.
TORC2 (Target of Rapamycin Complex 2) is rapamycin insensitive and consists of TOR2, Avo1,
Avo2, Avo3, Bit61 and Lst8. All studies on the cellular localization of TORC2 indicate that
TORC2 is at or near the plasma membrane. Its plasma membrane localization is essential for
viability, but TOR2 can also be found in the cellular interior.TORC2 is activated by direct
association with the ribosome and this mechanism ensures that complex is active only in growing
cells. The best-characterized and possibly the major TORC2 substrate is the protein kinase Ypk,
which is involved in actin polarisation, endocytosis and the most important in lipid biosynthesis.
In our studies we were trying to analyse the cross link between TOR2 complex, lipid metabolism
and DNA damage response. For this purpose we used analog-sensitive kinase allele Tor2 protein
(tor2as) which in presence of drug BEZ-235 acts like kinase-dead protein (lacks kinase activity).
We performed couple of spot assays including drug and hydroxyurea (commonly known agent
generating replication stress) treatment on tor2as strain. We have also visualised the actin filament
localisation in this strain to determine whether TORC2 can influence actin dynamics during
replication stress or not.
In this work, using lipid biosynthesis and checkpoint mutants of Saccharomyces cerevisiae we
were investigating the role of lipid metabolism in checkpoint activation and DNA damage
response.
Assays in a number of mammalian cancer cells have shown that mTOR inhibition acts
synergistically with various chemotherapeutic treatments. The information presented in this
poster may help in understanding of the pathway which links TORC2 inhibition to DNA damage
hypersensitivity which might be therapeutically relevant.
100
EFFECTS OF MUTATION IN CINR AND CINI GENES OF RHIZOBIUM
LEGUMINOSARUM BIOVAR TRIFOLII STRAIN 24.2
Kamila Rachwał, Anna Marzec, Jarosław Grządziel, Monika Janczarek
Department of Genetics and Microbiology, University of Maria Curie-Skłodowska, Akademicka 19 st.,Lublin 20-033,
Poland
Rhizobium leguminosarum is a gram – negative, motile, non-sporulating, nitrogen–fixing
bacterium, which can exist as a free-living bacterium, but also can live in the symbiotic
relationship with legumes (e.g.R. leguminosarum biovar trifolii has ability to associate with
plants of the genus Trifolium). Outer surface of rhizobial cells contains several polysaccharides,
which play a crucial role in the adaptation to both these habitats. One of these polymers,
exopolysaccharide (EPS) is involved in plant–microbe interactions, such as biofilm formation on
plant roots and forming special organs termed nodules. Inside nodules atmospheric nitrogen is
reduced to ammonia by the enzymatic complex of nitrogenase.
In Rhizobium leguminosarum bv. trifolii,cinI and cinR genes are engaged in quorum sensing
phenomenon, which consists of cell-density-dependent gene expression control, and involves the
production and detection of extracellular signaling molecules called autoinducers. Protein
products of cinI and cinR genes are components of AHL-based quorum sensing system called cin
responsible for the production of N-(3- hydroksy-7-cis-tetradeconyl)-L-homoseine lactone
(AHL), which serves a signaling molecule, in response to which CinR induces cinI gene. Third
element of the cin system, CinS is anti-repressor protein, that can enhance gene expression or
RNA levels. Quorum sensing system in R. leguminosarum affects EPS production, which plays a
very important role in this bacterium in successful colonization of new environments, biofilm
formation and establishment of effective symbiosis with respective host plants.
In this work, we performed several experiments using a site-directed mutagenesis in order to
obtain mutant strains with a mutation in the cinI and cinR genes, whose characteristics allows to
understand the role of these genes inR. leguminosarum bv. trifolii.
Acknowledgments: This work was supported by the grant from the National Centre of Science
no. DEC – 2012/07/B/NZ1/00099.
101
SYNTHESIS, STRUCTURAL CHARACTERIZATION AND BIOLOGICAL
STUDIES OF RUTHENIUM COMPLEXES WITH 2HYDROXYMETHYLBENZIMIDAZOLE
Patrycja Rogala1*, Agnieszka Jabłońska-Wawrzycka1, Grzegorz Czerwonka2, Maciej
Hodorowicz3, Barbara Barszcz1
1
Institute of Chemistry, Jan Kochanowski University, 15G Świętokrzyska Str., 25-406 Kielce, Poland,
*e-mail: patrycja.rogala@gmail.com
2
Department of Microbiology, Institute of Biology, Jan Kochanowski University, 15G Świętokrzyska Str., 25-406
Kielce, Poland,
3
Faculty of Chemistry, Jagiellonian University, 3 Ingardena Str., 30-060 Kraków, Poland
Ruthenium complexes have attracted much attention as possible building blocks for new
transition-metal-based antitumor agents. Some chemical properties of ruthenium complexes, such
as rate of ligand exchange, range of accessible oxidation states, and ability of ruthenium to mimic
iron in binding to certain biological molecules make these compounds well suited for medicinal
applications as an alternative to platinum antitumor drugs. Additionally, Ru(II) and Ru(III)
compounds can be used as metaloorganic drugs of many infectious diseases caused by parasites,
bacteria and fungi.
We therefore focused our studies on correlation between structure of ruthenium complexes in
higher oxidation states and their biological properties. The reactions of a mother solution of
RuCl3 with 2-hydroxymethylbenzimidazole (2-CH2OHBIm, L1) yielded two ruthenium
complexes: (HL1)2[RuVICl6]·2C2H5OH·4H2O (1) and (HL1)4[RuIVCl6]·2Cl·4H2O(2). The
obtained compounds have been characterized using elemental analysis, UV-Vis, IR
spectroscopies, magnetic study, and single crystal X-ray diffraction.We employed 2-CH2OHBIm
as ligand because it contain two potential donor sites, viz. to pyridine nitrogen and
hydroxymethyl oxygen atoms. This ligand has also biological activity: antibacterial, antifungal,
and antiviral activities.
Biological properties of ruthenium complexes were tested. Microtiter plate assay revealed that
ligand and ruthenium complexes reduced amount of Pseudomonas aeruginosa PAO1 biofilm.
However other studies showed only minimal inhibition of growth of P. aeruginosa PAO1
planktonic cells. Obtained data suggests that ruthenium complexes influence on first step of
biofilm formation process. It is connected with cells adhering to surface or reducing biofilm
matrix density. Another assays show that ruthenium complexes bind to double-strand DNA
(dsDNA) – chromosomal DNA from Haemophilus influenzae strain Rd. Ruthenium compounds
also act as PCR inhibitors where reduce amount of non-specific products (in concentration 40
µg/ml) and in higher concentrations (200 µg/ml) stop reaction. Binding to proteins will result in
weakening of the PCR efficiency reaction, not its specificity.To confirm this observation, DNA
binding assay was performed. The presence of the ruthenium complexes resulted an increase of
the peak intensity at λ = 260 nm which is specific for dsDNA. The experimental data suggests
that examined ruthenium complexes can bind to dsDNA rather than to proteins.
102
BRAINBOW AND CLARITY – NEW METHODS TO REVOLUTIONIZE
NEUROSCIENCE
Ewa Rojczyk-Gołębiewska1, Artur Pałasz1, Ryszard Wiaderkiewicz1
1
MedicalUniversity of Silesia, Departament of Histology and Embryology, Katowice, Poland
General anatomical structure and physiology of the brain is already well known. There are also
some tools for investigation of brain functions in different conditions and to modulate neural
activity. However, complexity of this organ is supposed to be connected with multitude of
synapses that are often not stable in time and space. This fact gave rise to connectomics – the
branch of neurobiology focusing on synapses and their high resolution imaging. Moreover, it is
important to visualize the whole brain and to trace specific types of neurons not only in thin
sections, but also in bigger blocks of tissue.
To head the challenge to image complicated neural networks accurately, two brand new methods
have been developed. One of them, called “Brainbow”, enables visualization of neurons and their
connections in extremely high resolution. General idea is to create genetic constructs with several
genes encoding fluorescent proteins flanked by multiple lox sites. In such contructs only genes
directly following the promoter can be expressed. Then, after creating mouse lines expressing
constructs, activated Cre recombinase excises/inverts DNA between one pair of lox sequences
which creates dozens of possible colour combinations.
The newly-introduced CLARITY metod should make possibile to visualize three-dimensional
structure of the translucent brain tissue using the hydrogel polymeric network. After hydrogeltissue hybridization, electric field is applied and the electrophoretic clearing takes place. As a
result, membrane lipids are extracted out of the tissue while fine-structure and crosslinked
biomolecules are leaved in place available for imaging and molecular phenotyping. This uniquely
original technique seems to be a milestone in the neuroscience creating a brand new viewpoints
completely different than standard glass slide immunostaining.
The image of mouse dentate gyrus
obtained with the use of “Brainbow”
The image of nervous system projections
in the intact Thy1-EYFP mouse brain
obtained with the use of CLARITY
103
DESIGN OF THE shRNA NUCLEOTIDE SEQUENCE AND LEADING
THREAD PREDICTION IN GENE THERAPY
Bożena Rolnik1 , Michał Jakubczak1, Marta Iwanaszko1
1
System Engineering Group, The Silesian University of Technology, Gliwice, Poland
Research on the regulation of genes expression and genes functionality using double-stranded
RNA (dsRNA) are carried out since 1990s. During this time it was proven that extracellular
constructs of RNA are able to create bindings with transcripts and block the translation process.
As the result the gene expression is stopped. This process is called RNA interference (RNAi).
There are a lot of specific dsRNA types with various characteristic, which are used in the
research. One of them is shRNA (short-hairpin RNA) - it is a short dsRNA with a small loop in
the one end. Using shRNAs in tests doesn't activate interpheron pathway, which is result of
introduction other RNA duplexes into cell. This solution decrease inflamation formation
probability, which is very useful in therapies.
RNAi process requires only one strand from dsRNA (called the leading strand), which works as
the probe for mRNA. The strand is choosen by Dicer nuclease and it is dependend on the WatsonCrick type bonds on the dsRNA structure end. Then, the strand is directed to the RISC complex.
This Dicer-dependend enzymatic mechanism increase the RNAi efficiency. Theoretically any
transcript sequence could be a target for shRNA.
In the following work we present results of student project, which consists of two main parts:
designing the nucleotide sequence of shRNA specific to selected target and predicting the leading
strand using molecular docking of shRNA to Dicer protein.
We have prepared a Matlab script, which uses commercial bioinformatics applications and
databases (GeneBank, Clustal, Blast) for finding the target sequence, doing multisequence
alignment and checking final sequence similarity to other known genes. The script generates the
target-complement sequence with chosen parameters, for example length and GC pair percentage.
It also adds a loop with random nucleotides.
In the second part we dock the final shRNA to the Dicer protein and calculate energies between
Dicer and both ends of the shRNA. This allow us to predict the leading strand. As the control set
for testing our solution we have used sequences from the research where the leading strand is
known.
In our work we used three genes and genes group significant in oncogenesis and cancer
development process. First is HIF (Hypoxia Induced Factor) gene, which is activated during
tumor creation. Product of this gene actives VEGF gene and the final result is angiogenesis.
Second is IKK genes - group of IκB inhibitors. Blocking translation of this group could cause
increased transcription of IκB and as a result decreased transcription of NFκB genes. NFκB is
responsible for antiapoptosis interaction and probably oncogenesis and cell cycle regulation. The
last gene is Mdm2 - inhibitor of proapoptotic protein p53. Increased expression level of p53
could cause incrased sensitivity for DNA demage by radiation.
104
ALTERATIONS OF MANGANESE AND CADMIUM TRANSPORT IN
HUNTINGTON’S DISEASE
Aleksandra Różycka1, Monika Szeliga2, Jan Albrecht2
1
Warsaw University of Life Sciences, Faculty of Horticulture, Biotechnology and Landscape Architecture,
Interfaculty Biotechnology Division, 159 Nowoursynowska Street, 02-776 Warsaw
2
Polish Academy of Sciences, Mossakowski Medical Research Centre, Department of Neurotoxicology, 5
Pawinskiego Street, 02-106 Warsaw
Huntington's Disease (HD) is inherited brain disorder, which destroys cells in the basal ganglia,
the part of the brain that controls emotion, movement, and cognitive ability. HD is caused by a
mutation in a HTT gene, which is located on chromosome 4. People with HD have an abnormally
high number (approximately 40 or more) of CAG triplets encoding glutamine.
Analysis of metal neurotoxicity has revealed similarities in the pathophysiological mechanisms of
HD and other neurodegenerative disorders. Both protein aggregation and oxidative stress, have
been implicated in the toxicity associated with HD and metal overexposure. Studies on mouse
model of HD revealed that one of the features of cells with mutated HTT gene is an altered Mn2+
uptake and a simultaneous increase of intracellular accumulation of Cd2+, which are highly
neurotoxic metal.
In this project we try to verify hypothesis that the observed alterations in level of Mn2+ and Cd2+
result from the altered expression and/or activity of proteins engaged in the transport of these
metals, that is: transferrin (TF), its receptor, TfR, divalent metal transporter 1 (DMT1) and ZIP8
protein.
105
THE INFLUENCE OF SODIUM CHLORINE ON SYNTHETIC
ESTROGENS UTILIZATION BY SELECTED FILAMENTOUS FUNGI
Anna Różycka1, Piotr Michnicki1, Sylwia Różalska2, Jerzy Długoński2
1
Biotechnology, University of Łódź, Poland
2
Introduction: Endocrine disrupting chemicals (EDCs) are toxic compounds of anthropogenic
origin which have ability to interfere with the endocrine systems of animals. These pollutants
have influence on many physiological functions of aquatic organisms. An example of EDCs are
mestranol (MES) and ethinylestradiol (EE2) which are commonly used in oral contraceptive pills.
These compounds can be detected also in the areas.The removal of xenobiotic substances by
microorganisms (including filamentous fungi) is a very efficient method, but a special interest
should be noticed on utilization in the presence of substances which have impact on microbial
activity (e.g. salinity).
Aims: In this work we assessed the utilization of several filamentous fungi for mestranol and
ethinylestradiol. Moreover the removal of investigated EDCs in the presence of salinity was
checked.
Materials and methods: Chosen strains of filamentous fungi were cultivated on Lobos medium
with EE2 or MES (10 mg/l), and with or without addiction of NaCl. Different concentration of
salinity were used: 0.8;1,8; or 2.8%. The samples were extracted after 72 h of incubation with
using QUECHERS method and analyzed by HPLC MS/MS.
Result: The obtained results show that among studied 45 fungal strains, 12 of them were able to
utilize EE2 and 3 used MES in range 90%. In the presence of salinity (0.8 or 1.8 or 2.8%) the
most efficient degradation of EE2 was observed for the strains IM 6324 and Aspergillus
versicolor IM 2161. The EE2 utilization conducted by the strain IM 6324 did not differ in the
presence or absence of NaCl. The EDCs utilization by the A. versicolor IM 2161 was slightly
inhibited (in 15%) only in the presence of 2.8% of NaCl.
Conclusion: We assessed that two selected strains were able to utilize one of the EDCs in
presence of salinity without decrease of efficiency.
106
CYTOTOXICITY OF THIOSUGARS IN CANCER CELL LINES
Joanna Sarnik1, Anna Czubatka1, Zbigniew J. Witczak2, Tomasz Popławski1
1
Katedra Genetyki Molekularnej Uniwersytetu Łódzkiego; ul. Pomorska 141/143 93-236 Łódź
2
Department of Pharmaceutical Sciences, Nesbitt School of Pharmacy,Wilkes University, Wilkes-Barre, PA 18766,
Thiosugars belong to the group of chemical compounds containing sulfur atom. Thiosugars
possess biological activities because of the presence of sulfur atom which differs from their
oxygen analogs. These differences result from the conformation, flexibility and geometry of these
two groups of compounds. The introduction of sulfur atom into the structure of sugars may
prevent their susceptibility to enzymatic or chemical degradation. Besides naturally thiosugars
such as those found in marine sponge Clathria pyramida, many of them are synthetic.
The aim of our project was to compare the cytotoxicity of synthetic thiosugar denoted T VI
(Ryc.1) and T VII (Ryc.2). T VII has four hydroxyl groups in the carbon ring and in T VI
analogue these groups are blocked by acetylic groups. The cytotoxicityof these thiosugars was
tested on four cell lines: human cervix adenocarcinoma (HeLa), human breast adenocarcinoma
(MCF7), human lung adenocarcinoma epithelial (A549) and human colon adencarcinoma
(LoVo). Thiosugar cytotoxicity was determined by colorometric assay (Cell Counting Kit-8).
All experiments show us that thiosugars are cytotoxic for all studied cancer cell lines. We
observed that T VI was more cytotoxic than T VII. For both thiosugars, the concentration above
1,1 mM caused lethal effect – viability below 50% in all studied cell lines. The most sensitive
cancer cell line to thiosugars VI and VII was MCF7 since 69 µM of T VI and 550µM of VII were
lethal – viability below 50% for this cell line.
T VI which has four hydroxyl groups in the carbon ring blocked by acetylic groups is more
cytotoxic than T VII which has four hydroxyl groups in carbon ring. The most promising
candidate for anticancer treatment, among studies thiosugars, for further research is T VI, which
proved to be the cytotoxic in micromolar concentration for breast cancer cell line.
This work was supported
2011/01/B/NZ4/03391
Ryc.1TVI
by
the
National
Science
Centre,
decision
No.DEC-
Ryc.2 T VII
107
EXPRESSION AND PURIFICATION OF THERMOSTABLE DNA
POLYMERASES PFU AND TAQ
Kajetan Sawa1,2, Monika Filipiak1,2, Weronika Serafin1,2, Dawid Żyła1,2,3
1
Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Kraków
2
Scientific Association of Biotechnology Students „Mygen”
3
Department of Physical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian
University, Kraków
Thermostable DNA polymerases are widely used in molecular biology since their discovery in
1976, when Taq polymerase was isolated from thermophilic bacterium Thermus aquaticus. Taq
revolutionized molecular biology allowing fast and easy DNA multiplication in the polymerase
chain reaction (PCR), which was previously troublesome and time-consuming. Further
discoveries introduced DNA polymerase isolated from archaeon, Pyrococcus furiosus, by
Lundberg. In the research superiority of Pfu over Taq was clearly shown. However it has smaller
pace, the most-high fidelity of reaction product is the reason why Pfu is often chosen over Taq,
especially in criminal cases and other instances requiring high precision from enzymes.
The target of the Scientific Association work was to acquire active thermostable polymerases at
as low cost as possible. Project aimed an overexpression and purification of Taq and Pfu
polymerases in expression strain BL21 DE3 pLys. Using thermostability feature of both
polymerases first step of purification was applied. Further steps involved ammonium sulphate
precipitation and dialysis to storage buffer. After purification, optimization in comparison to
commercially available enzyme was done, as well as assay of the thermostability of protein. All
of this was done to measure the quality of product and find possible room for enhancement in
future.
108
TWO-DIMENSIONAL GEL ELECTROPHORESIS IMAGE ANALYSIS IN
DIFFERENTIAL GEL-BASED PROTEOMICS
Marta Seczyńska
Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow
Techniques of gel-based proteomics such as conventional two dimensional gel electrophoresis (2DE) or difference gel electrophoresis (DIGE) still remain popular and powerful tools for
identification of proteins that are differentially expressed across treatment condition or in
different sample types (e.g healthy/diseased, wild type/knockout). Due to the overwhelming
number of proteins separated on each gel and significant variation among gel images, the analysis
of 2-DE data is challenging. Having direct impact on obtained qualitative and quantitative results,
this crucial step in proteomic workflow requires appopriate software package. There are many
comercially viable tools such as ImageMaster, Decyder (GE Healthcare), PDQuest (Bio-Rad) or
Delta2D (Decodon) which uses a very similar scheme of analysis, sometimes achieved by
different algorithms.
Major steps in gel image analysis involve data aquisition, spot detection, spot quantification, gel
matching and statistical analysis. Each of them can be a source of mistakes which propagated
lead to drawing wrong biological conclusions. However, in contrast to the part of proteomic
experiment which takes place in laboratory, little attention is paid to this data analysis issue.
Therefore, the aim of the presented poster is to emphasize importance of the careful investigation
of post-experimental process as well as highlight good practices and challenges. Moreover, it will
be also shown that all software packages require extensive, time-consuming manual editing and
correction. Such user intervention may reduces objectivity and reproducibility.
Although gel-free methods slowly dominates proteomics, gel-based methods are still methods of
choice in many laboratories, then improving the 2-DE and DIGE images analysis is crucial to
receive comparable and reliable results.
109
NANOCELLULOSE- POLYMER OF THE FUTURE
Agata Seliga1
1
Lodz University of Technology, Faculty of Biotechnology and Food Sciences, ul. Wólczańska 171/173, 90-924
Łódź, Poland Scientific Association of Biotechnology Students „Ferment‟
The rapid growth of interest in the nanomaterialspromotes the pursuit of technology of
nanostructures from commercially available materials.
Cellulose is one of the most accessible and low-cost organic polymers on the market. Special
properties of cellulose nanofibers (large surface area, high chemical reactivity; interesting optical
properties, electrical and magnetic properties, a high level of biodegradation) make this polymer
useful in the textile, paper, medical and cosmetic industries. In order to isolate the cellulose of
plant biomass and its fragmentation into nanopolimer physical, chemical or biological pretreatment of raw material is carried out. Processing of biomass should effectively remove the
materials surrounding from the cellulose: lignin and hemicellulose and have no negative impact
on it.
Figure 1. Nanocellulose production.
Nanocellulose is a polymer with a high potential for industrial implementation. The choice of
efficient, cost-effective and environmentally friendly method of its obtainment would result in
opening of new opportunities for the use of agricultural waste.
REFERENCES:
[1] Wąchała R., Ramięga T., Pyć R., Antczak T.: Otrzymywanie włókien nanocelulozy,
Biotechnology and Food Science 75(2), 2011, 87-100.
[2] Szczęsna-Anczak M., Kazimierczak J., Antczak T.: Nanotechnology - Methods of
Manufacturing Cellulose Nanofibres, Fibres and Textiles, 91(2), 2012, 8-12.
[3]
Nanoscale
investigation
http://www.nanowerk.com
advances
bioethanol
production
from
woods.:
110
ELICITATION AND THE PHENYLOPROPANOIDS PRECURSOR
FEEDING AS TOOLS FOR IMPROVING NUTRACEUTICAL
POTENTIAL OFLENS CULINARISSPROUTS
Łukasz Sęczyk1, Michał Świeca2, Urszula Gawlik-Dziki2
1
Student Scientific Circle of Biochemistry and Food Chemistry
2
Department of Biochemistry and Food Chemistry, University of Life Sciences, Skromna Str. 8, 20-704 Lublin,
Poland
Elicitation (the process based on natural plant defense system) allow to induced and/or expanded
the biosynthesis of secondary metabolites. Additionally, the effectiveness of this action may be
enhance by precursor feeding (providing exogenous precursor for biosynthesis of final product).
The effect of elicitation with UV lightand/ or L-phenylalanineprecursor feedingon induction the
phenylopropanoids metabolism and antioxidant activity of lentil sprouts were studied. For this
purpose phenylalanine-ammonia lyase (PAL) activity, total phenolics and flavaonoids
content,free radical scavenging ability and reducing power were determined. Four types of
samples were prepared: control (C), UV light treated (UV), phenylalanine feeding(Phe) and
exposed to both factors (Phe+UV).
All studied germination condition (phenylalanine feeding, UV treatment and elicitation
andphenylalanine feeding and following UV treatment)led to change the phenylopropanoid
metabolism of lentil sprouts, these changes were translated into the increased phenolics content
and antioxidant activity.The greatest impact on PAL activity had the combined effect of precursor
feeding and elicitation (Phe+UV), where an increase by 57% compared with control was
observed.Furthermore,used techniques significantly increasedthe total phenolics content (by
about 10% for each samples) and the total flavonoids content (from 26% (UV) to 46% (Phe)).
Phenylalanine supplementation led to obtain lentil sprouts with the highest antioxidant potential
(an increase by 26,5% (free radical scavenging ability) and 24,8% (reducing power)in
comparison to control).
In the light of results the studies, the used biotechnological approaches may be the useful
techniques for inducing the phenylpropanoid metabolism. Moreover, their action can lead to
improve content of natural antioxidants (phenolics) in non-processed food and consequently to
increase its potential health benefits.
111
CHANGING IN MEMBRANE LIPIDS OFPENICILLIUM CHRYSOGENUM
IN THE PRESENCE OF TRIBUTYLTIN CHLORIDE -A REACTIVE
OXYGEN SPECIES (ROS) INDUCER
Paulina Siewiera1, Marta Dudzik2, Przemysław Bernat, Adrian Soboń, Jerzy Długoński
1, 2
Department of Industrial Microbiology and Biotechnology, Faculty of Biology and Environmental Protection,
University of Łódź, Poland
Tributyltin (TBT) belongs to the group of organotins, which are commonly used
organometallicbiocides. This compound was globally employed as an ingredient of antifouling
paints and preservatives for wood, paper and fabrics. Special importance for the environment has
a high toxicity of tributyltin chloride. TBT is suggested to stimulate the generation of reactive
oxygen species (ROS) inside cells. One of the mechanism of toxicity is also the disruption of the
structure and function of biological membranes. Although TBT is toxic to microorganism, some
strains such as Penicillium chrysogenum, are capable of survival in the presence of high
concentrations of this compound.
A sensitive approach based on electrospray ionization tandem mass spectrometry has been
employed to profile membrane lipid molecular species in the Penicillium chrysogenum
undergoing TBT stress.
P. chrysogenumwas incubated in Sabouraud medium with TBT (10 mg/l) or without the biocide.
Bacterial biomass from the stationary phase of growth were used for the extraction of
phospholipids using the method of Folch. To create the phospholipid profiles of strain,high
performance liquid chromatography (HPLC)-MS/MS was applied, than individual phospholipids
were quantified by a multiple reaction monitoring method (HPLC-MRM).
A total of 38 phospholipid species were profiled quantitatively, using the LC/ESI/MS/MS
procedure. Under the influence of TBT content of phosphatidic acid (PA) and
phosphatidyloinositol (PI) increased, while levels of phosphatidylethanolamine (PE),
phosphatidylcholine (PC) and phosphatidyloserine (PS) decreased.
The great loss of PC and increase in PA in P. chrysogenum as compared with control, may be
responsible for destabilizing membrane bilayer structure, resulting in increase of its
permeability.It might also suggest thatthere were some connections between PA and ROS
signaling in P. chrysogenum exposed to TBT.
112
RELEVANCE OF CITRULLINATION IN PROTEIN FUNCTIONS AND
PATHOLOGICAL CONDITIONS
Agnieszka Sitarska, Karolina Płaza, Tomasz Kantyka, Jan Potempa
Department of Microbiology, Faculty of Biophysics, Biochemistry and Biotechnology, Jagiellonian University,
Krakow, Poland
Citrullination is a post-translational modification of proteins, catalyzed by the family of
mammalian enzymes – Peptidylarginine Deiminases (PADs). This Ca2+ dependent group consists
of five tissue-specific enzymes. They modify protein targets in epidermis (PAD1), skeletal
muscles, brain and secretory glands (PAD2), hair follicles (PAD3), white blood cells (PAD4) and
ovum (PAD6). PAD4 has a presumable role in cancer development. Dysfunction of PADs also
correlates with pathological conditions.
Citrullination leads to the conversion of arginine to a non-DNA encoded aminoacid citrulline by
side chain deimination.Loss of positive charge may alter the tertiary structure of the protein and,
as a consequence, affect its function.
Figure 1: Citrullination scheme derived from Mohanan et al. Potential Role of Peptidylarginine Deiminase Enzymes
and Protein Citrullination in Cancer Pathogenesis, Biochemistry Research International, vol. 2012
A number of proteins hold biologically relevant arginine residues exposed to their cleavage sites
or present in reactive loops. This applies to both coagulation cascade and complement system,
where proteolytic activation involves cleavage after arginine residue and regulation of the process
is performed by P1-Arg-containing inhibitors.
Lympho-epithelial Kazal-type-related Inhibitor (LEKTI) may also act as an example. It is a
multidomain serine proteases inhibitor secreted in epidermis and responsible for desquamation
control. Most of its fifteen domains hold arginine in P1 position which determines target
specificity. Citrullination may alter that characteristics therefore could be regarded as a regulatory
mechanism in desquamation process.
It has been proved that citrullinated proteins are targets for inflammatory response in autoimmune
diseases, eg. Rheumatoid Arthritis (RA). Elevated levels of antibodies against deiminated forms
of fibrinogen, vimentin, collagen II and α-enolase are present in peripheral blood and act as
diagnostic marker of RA.
113
Microenvironmental regulation of tumor progression and metastasis
Katarzyna Sitarz1
1
Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology; Scientific Association of
Biotechnology Students “Mygen”
Theoretical poster based on the article entitled “Microenvironmental regulation of tumor
progression and metastasis” which was published in Nature Medicine in November 2013.
Stromal cell types within the tumor microenvironment (TME) play a very important role in the
development of cancer. They raise hopes for researchers who are looking for an effective cancer
treatment. Their main advantage is the genetic stability which reduces the risk of resistance and
tumor reoccurrence. But we still have very few details about the influence of concrete factors in
TME for the development of cancer. Recent research has shown that the microenvironment of
cancer is capable of normalizing tumor cells. It stems from the fact that possible re-education of
stromal cells can be a good way to combat cancer. The poster will be an attempt to present the
roles of the TME during specific stages of cancer progression and metastasis, as well as recent
attempts to re-educate stromal cells within the TME to have anti-tumorigenic effects.
114
PHENOLIC COMPOUNDS IN SELECTED EDIBLE FOREST FRUITS
AND THEIR ANTIOXIDANT PROPERTIES
Agata Nowogórska1*, Monika Skwarek1, Adrian Witczak2, Jacek Patykowski1
1
Department of Plant Physiology and Biochemistry, Faculty of Biology and Environmental Protection, University of
Łódź, *nowogorska.agata@gmail.com
2
Institute of Forest Sciences, University of Łódź, Branch in Tomaszów Mazowiecki
Oxidative stress is a disturbance in the balance of redox reactions in favour of the prooxidant
ones. Damage caused by too large amount of reactive oxygen species and free radicals can be the
cause of many diseases, including neurodegenerative diseases, cancer, heart diseases and
atherosclerosis. Therefore, more and more attention is focused on a varied diet with various
compounds with antioxidant properties.
A good example of the rich sources of antioxidants are products of plant origin. Such properties
are characterized by low molecular compounds such as ascorbic acid and alpha tocopherol,
carotenoids and various phenolic compounds. The latter are represented by many phenol
derivatives. They include simple phenols (including phenolic acids) and their derivatives such as
flavonoids, isoflavones and hydrolysing or condensed tannins.
Selected forest fruits used by humans for thousands of years for food purposes were material for
experiment. Fruits of sea buckthorn (Hippophae rhamnoides L.) and wild rose (Rosa canina L.)
are a component of many dietary supplements, mainly because they are a rich source of vitamin
C. Blackberry (Rubus caesius L.) and elderberry (Sambucus nigra L.) fruits are recommended for
people with impaired immunity and during increased incidence of flu and cold, while blackthorn
(Prunus spinosa L.) fruits are a good ingredient for home made liquors.
The aim of this study was to determine the total antioxidant potential and the total phenolic
content of extracts of chosen fruits. It was interesting to verify the relationship between these two
parameters. Also concentration of flavonoids and phenolic acids, the representatives of the main
groups of phenolic compounds, were measured. Determination the group which constituted the
majority of total phenols was of particular interest.
The results showed that high total phenolic content was clearly correlated with high antioxidant
potential of the tested extracts. The highest values for both parameters were observed for extracts
of wild rose. Blackthorn fruit extract showed the second highest values of the above parameters
and also was characterized by high levels of phenolic acids and flavonoids. The highest content
of flavonoids was measured for elderberry extract.
In conclusion, the forest fruits are a rich source of compounds with health promoting properties.
A number of studies showed that such characteristics were demonstrated mainly in plants
growing in cold climate with short vegetation period. Also a higher content of phenolic
compounds was proved for alcoholic extracts as compared to water extracts. The fundamental
values of these compounds are based on the direct reactions with free radicals, scavenging
reactive oxygen species and prooxidative metal chelation.
115
PHENOLIC COMPOUNDS IN SELECTED POISONOUS FOREST FRUITS
Monika Skwarek1*, Adrian Witczak2 , Agata Nowogórska1, Jacek Patykowski1
1
Łódź, *monika.skwarek1988@gmail.com
2
Institute of Forest Science, University of Łódź, Branch in Tomaszów Mazowiecki
Forest fruits are a rich source of food for humans and animals. Some forest plants contain
compounds which are attractive for animals. However, some fruits have toxic compounds which
protect plants against herbivores.Poisonous plants can be often identified by unpleasant smell and
pungent taste. Animals generally recognize poisonous plants and avoid them. The susceptibility
or resistance to each plant poison are individual in both humans and animals. Among herbivores
there are invertebrateswhich live in the forest undergrowth. Their digestive tract is adapted to
metabolize toxic substances contained in the forest fruits.
The toxic properties of plant species are determined due to the presence of their active
substances. Toxic compounds in forest fruits include alkaloids, glycosides and phenolic
compounds. Among the latter there are simple phenols and their derivatives such as flavonoids,
isoflavones and hydrolysing or condensed tannins.
The aim of this study was to determine the total antioxidant potential of extracts of selected
fruits.The analysis of the impact of the total phenolic compoundcontent on the antioxidant
properties was also examined.In the tested samples the contents of phenolic acids and flavonoids
were also determined.The list of poisonous plants which occur in our climatic zone is very long.
Selected poisonous forest fruits such as privet (Ligustrum vulgareL.), lilly- of-the-valley
(Convallaria maialisL.),viburnum (Viburnum opulusL.),buckthorn (Rhamnus cathartica L.)and
poisonberry (Solanum dulcamaraL.) were used for the experiment.
The results showed that the high total phenolic content was related to high antioxidant potential
of the tested samples. The highest values for both parameters were observed for fruits of privet
and buckthorn. The highest content of phenolic acid was measured for privet fruits while the
highest content of flavonoids for buckthorn fruits. The lowest values of total phenolic content and
antioxidant potential were determined in lilly-of-the-valley fruits.
In conclusion, the investigated plants were characterized by high levels of antioxidant potential
and high content of phenolic compounds. However,the presence of compounds such as
glycosides and alkaloids in plants can make them poisonous to living organisms.
116
A NEW METHOD OF IDENTIFYING PATHOGENS – LAMP AS
COST-EFFECTIVE AND RAPID ALTERNATIVE FOR PCR
Alicja Smolarz1, Daria Białasik2
1,2
Poznań University of Live Sciences
Loop-mediated Isothermal Amplification ("LAMP") is a cost-effective, simple, rapid and
specific nucleic acid amplification method. It relies on use of 4 different primers specifically
designed to recognize 6 distinct regions on the investigate gene and the reaction process proceeds
at a constant temperature using strand displacement reaction. Amplification of target gene can be
completed in a single step, by incubating the mixture of samples, primers, DNA polymerase with
strand displacement activity and substrates at a constant temperature (about 65°C). Detection
requires special apparatus what can be disadvantage. LAMP provides high amplification
efficiency, with DNA being amplified 109-1010 times in 15-60 minutes. Because of its high
specificity, the presence of amplified product can indicate the presence of investigate gene what
is very important for instance in pathogen detection.
ADVANTAGES:
•There is no need for a step to denature double stranded into a single stranded form.
•The whole amplification reaction takes place continuously under isothermal
conditions.
•The amplification efficiency is extremely high.
•By designing 4 primers to recognize 6 distinct regions, the LAMP method is able to
specifically amplify the target gene.
•The total cost can be reduced, as LAMP does not require special reagents.
•Amplification can be done with RNA templates following the same procedure as with
DNA templates, simply through the addition of reverse transcriptase.
Starting structure for LAMP cycling
REFERENCES:
[1] Adam Burzyński, Marta Jankowska „Amplifikacja in vitro wybranych odcinków DNA i RNA
metodą Loop-mediated isothermal AMPlification (LAMP)” Poznań, 2012
[2]http://loopamp.eiken.co.jp/e/lamp/
[3] http://nar.oxfordjournals.org/content/20/7/1691.abstract
117
Qualitative analysis of the products of microbial biodegradation of 4-nnonylphenol using GC-MS
Adrian Soboń, Milena Adela Piątek, Sylwia Różalska, Jerzy Długoński
Faculty of Biology and Environmental Protection, Department of Industrial Microbiology and Biotechnology,
University of Lodz email: asobon@biol.uni.lodz.pl
Mass spectrometry techniques are widely used in the qualitative and quantitative analysis of a
wide range of substances. The combination of mass detection with chromatographic techniques
such as gas chromatography allows to perform complex experiments which provide to obtain a
specific information about the structure or the concentrations of the substances in complex
matrices.
The presented work is focused on the use of the Agilent 7890 GC coupled with an Agilent 5975C
mass detector with EI ion source to find and identify the products of microbial biodegradation of
xenobiotics. Determination of the characteristic fragment ions from the test substance was based
using Extracted Ion Chromatogram mode (EIC). The proposed methodology allows filtering of
signals leading to the analysis of the signals coming only from potential metabolites of the
compound.
EIC mode was used for qualitative analysis of biodegradation 4-n-nonylphenol (4-n-NP) in
submerged culture of the filamentous fungus Metarhizium robertsii IM 6519. This strain is
capable to degradation >90% of 4-n-NP (50 mg/l) during 72 h of incubation. Additionally,
28 products of the xenobiotic biodegradation were identified. Analyzed compounds containing
singly or doubly hydroxylated aromatic ring and hydroxyl group in different positions of the
linear chain. Moreover, several metabolites with carboxyl group in the end of the alkan chain
were found. Among this metabolites the most prevalent were compounds with hydroxylation at
the C1 position of aliphatic chain. The fungi also removes carbons from the linear chain of
hydroxylated metabolites. Analysis results with the interpretation of mass spectra for selected
metabolites will be shown in presented poster.
The work was supported by the National Center of Science, Poland (Project No. UMO2011/01/B/NZ9/02898).
118
NEXT-GENERATION SEQUENCING – THE FUTURE OF MOLECULAR
BIOLOGY
Joanna Stachecka, Mateusz Chrzanowski
Poznan University of Life Sciences, Faculty of Agriculture and Bioengineering, KNSB Operon, Wojska Polskiego 28,
60-637 Poznan, e-mail: joanna.stachecka@gmail.com
Invention of the sequencing technologies have been a milestone in molecular biology. Those
techniques allow us to learn the nucleotides sequence of PCR products, which has an essential
meaning in fields like phylogenetics, taxonomy, population genetics, genomics, etc.
Nowadays there are multiple of sequencing methods, but for the last thirty years the most popular
is chain termination method developed by Frederick Sanger in 1977. The biggest disadvantage of
Sanger‟s method are the enormous costs. At a time when most of the molecular researches use
nucleotide sequences the need of cheaper techniques has arise. In a response to this need
scientists develop new methods which are based on different mechanisms. Their advantages are
much lower costs and the maintenance of Sanger‟s method accuracy. They are called
the next-generation sequencing methods. Among them the most promising are: single-molecule
real-time sequencing (SMRT), ion semiconductor sequencing, 454 pyrosequencing, Illumina dye
sequencing, SOLiD sequencing.
In our poster we present short characteristic of techniques listed above and compare them in
terms of costs, time requirements and accuracy. We selected the leading one – single-molecule
real-time sequencing (SMRT) – gave detailed description and compared to the Sanger‟s method.
We believe that constant advancement of next-generation sequencing methods and invention of
new techniques will finally lead to inexpensive, quick and accurate method, that would be an
alternative to expensive Sanger‟s method.
119
THE NOVEL INHIBITOR OF THIOREDOXIN- THIOREDOXIN
REDUCTASE SYSTEM
Joanna Stachura1,2, Szymon Kłossowski3, Angelika Muchowicz1
1
Department of Immunology, Centre of Biostructure Research, Medical University of Warsaw, Warsaw, Poland
2
Warsaw University of Life Sciences, Warsaw, Poland
3
Institute of Organic Chemistry, Polish Academy of Science, Warsaw, Poland
Thioredoxin- thioredoxin reductase system consists of multifunctional and ubiquitous proteins. It
has been proven that this enzymatic system takes part in protection cells from oxidative stress,
involves in process of proliferation and inhibits cancer cells apoptosis. What is important,
increase level of Trx- TrxR system proteins has been observe in tumor formation and
angiogenesis. Thioredoxin affects reduction of oxidized protein disulfide bond to sulfhydryl
groups, which provides cells to change of redox state. It is suggested that effect of antitumor
therapy could be increased by the inhibition of this enzymatic system.
The aim of the study was to investigate the mechanism of action of new thioredoxin inhibitor.
The presence of monomeric or dimeric forms of Trx has been evaluated in two ways: in in vitro
studies by Western Blotting technique and by SDS- PAGE with use of recombinant thioredoxin.
The in vitro studies have been conducted on cancer cell lines: Raji (human Burkitt‟s lymphoma)
and NB4 (human promyelocytic leukaemia). The activity of enzyme has been studied with
insulin reduction assay. The cytostatic/cytotoxic effect of SK053 has been analyzed with MTT
test.
It appears that SK053 binds to the enzyme cysteins in active center and prevents the creation of
reduced form of Trx. This mechanism leads to decline in formation of thioredoxin dimmers in
recombinant Trx as well as in cancer cells. Furthermore after incubation cancer cells with
inhibitor the lower activity of Trx- TrxR system was observed. SK053 shows the
cytostatic/cytotoxic effect against tumor cells.
It is believed that cancer cells protect themselves from oxidative stress or apoptosis by increased
production of proteins of the thioredoxin- thioredoxin reductase system. The reduced form of
thioredoxin has the ability to inhibit theapoptosis. SK053 is an effective inhibitor of this
enzymatic system. Further studies are needed to evaluate the antitumor effectiveness of this
inhibitor.
References:
Kłossowski Sz, Muchowicz A, Firczuk M, Świech M, Redzej A, Gołąb J, Ostaszewski R. Studies
toward Novel Peptidomimetic inhibitors of Thioredoxin- Thioredoxin Reductase System. J Med
Chem. 2012 Jan 12;55(1):55-67. doi: 10.1021/jm201359d.
120
Bcl-2 – HOPE IN THE BATTLE AGAINST CANCER?
Daria Stapurewicz1
1
Scientific Student`s Group for Biotechnology; University of Warmia and Mazury in Olsztyn; Faculty of Biology and
Biotechnology; Supervisor: dr hab. Iwona Bogacka, prof. UWM
The term apoptosis comes from Greek and means “a leaf falling down the tree”. It is a natural
process of the programmed cell death. A significant role of apoptosis is to maintain the tissue
homeostasis. It acts to eliminate damaged cells that may cause undesirable effects.
The major function of B-cell lymphoma-2 protein family (Bcl-2) is the regulation of apoptosis
processes. Bcl-2 family divides into three subfamilies, anti-apoptotic, pro-apoptotic and “BH3
only”. Each of them consists at least one α-helical homologous BH (BH1, BH2, BH3, BH4)
domain. Anti-apoptotic proteins, i.e. Bcl-2, Mcl-1, usually have four α-helical domains (BH1 to
BH4). Bak, Bax, Bok or Bid belong to pro-apoptotic proteins and have BH1, BH2 and BH3
domains. BH3 proteins have only BH3 domain. BH1 and BH2 domains are responsible for the
formation of canals in the mitochondrial membrane, as well as regulation of the cytochrome c
release. These two domains may act as pro-apoptotic and/or anti-apoptotic factors. On the other
hand, BH3 domain stimulates apoptosis. The classification of Bcl-2 proteins is not clear due to
the fact, that changes in environmental conditions may alter proteins phenotype.
Disturbances in apoptotic processes may lead to uncontrolled division of cells. These events take
place during carcinogenesis. Tumor cells have mechanisms protecting them from programmed
cell death. It is well documented, that the high level of Bcl-2 proteins expression occurs in most
cancer cases. Both in vivo and in vitro experiments suggest that the high expression of Bcl-2 can
be responsible for radiotherapy and chemotherapy. On the other hand, blocking the anti-apoptotic
proteins expression (Bcl-2 and Bcl-xL) may increase cellular sensitivity for chemotherapeutic
drugs.
There is increasing amount of evidence showing that Bcl-2 proteins might serve as markers for
many diseases. In the light of the existing knowledge, it seems to be important to focus studies on
the use of Bcl-2 proteins for diagnostic and therapeutic purposes.
121
ANALYSIS THE DETOXIFICATION PROPERTIES OF PRODUCTS
FROM THE GROUP "DetoxPad"
Marcin Staśko, Adriana Żyła
Faculty of Chemistry – University of Opole
Too often we hear about heavy metals and toxins polluting our environment and our bodies,
which can cause many ailments in our organisms. Many pharmaceutical companies and similar
facilities are coming out to our needs with introducing many doubtful medicines and
prophylactics. One of these products are detox foot patches (pads).
The question is: does detox food pad patched to our skin really has ability of cleaning our body
and can heal us from illness caused by toxins and heavy metals? Why does foot pads change their
colors, and why suddenly they stop? Do they really work? We will try to answer that questions,
by carrying out scientific tests.
122
POTENTIAL APLICATIONS OF IMMOBILIZED LACTOSE IN DAIRY
INDUSTRIES
Magdalena Stobińska1, Dawid Urbański2
1,2
Center of Bioimmobilisation and Innovative Packing Materials, West Pomeranian University of Technology,
Szczecin
1
mstobinska@zut.edu.pl
The enzyme β-galactosidase, most commonly known as lactase, which hydrolyses lactose into its
monomers that is glucose and galactose has potential applications in food processing industry.
Treatment of milk and milk products with lactase to reduce their lactose content seems to be an
appropriate method to increase their potential uses and to deal with the problems of lactose
insolubility and lack of sweetness.
The application of β-galactosidase in the hydrolysis of lactose in dairy industry has attracted the
attention of researchers. Although most industries still hydrolyze lactose use free enzyme, but the
immobilization of β-galactosidase is an area of great interest because of its potential benefits. The
use of immobilization technology is of significant importance from economic point of view since
it makes reutilization of the enzyme and continuous operation possible and also precludes the
need to separate the cells from the whey following processing. It can also help to improve the
enzyme stability. Nowadays, immobilized β-galactosidase is intensively being used in lactose
hydrolysis of milk/whey and has been tested for the production of galactooligosaccharides.
One of the enzymes immobilization methods is microencapsulation. Microcapsule consists of
liquid core and polymer shell. Alginate gels beads can be easily separated from the reaction
medium by filtration and can be used many times.
The aim of this study was to evaluate the possible application of the microcapsules using a coextrusion technique. Liquid core (e.g. solution of enzyme) and shell hydrogel materials (e.g.
aqueous solution of sodium alginate) are pressed through concentric nozzle, with the core
material in the central nozzle, and the shell material in the outer one. A compound drop
composed of a droplet of core fluid encased by a layer of shell fluid forms. The shell is then
hardened by appropriate means, by chemical crosslinking in the case of alginate in gelling bath
with calcium chloride.
Microencapsulated lactase within the alginate beads allows to be separated from the reaction
solution and can be used potentially many times which leads to reduction of cost and formation
of bioconversion solution without enzyme. In the same time it was found that immobilized
enzymes exhibit greater activity and stability during the bioconversion process and after during
long-term storage. The obtained results confirm that novel alginate core/shell microcapsules with
lactase in liquid-core could contribute to the development of new continuous processes.
Results of performed experiments show that in relation to the different type of immobilization
enzyme, the liquid core microcapsules are promising possibilities to be applied at industrial scale.
123
REVIEW OF IMMOBILIZATION METHOD FOR MICROORGANISM
Magdalena Stobińska1 , Dawid Urbański2
1,2
Center of Bioimmobilisation and Innovative Packing Materials, West Pomeranian University of Technology,
Szczecin
1
mstobinska@zut.edu.pl
In biotechnology immobilization is use for protection to the cell from unfavourable conditions.
This can provide increased resistance to changes in conditions such as pH or temperature.
Immobilization methods may be classified into three main categories: immobilization on carrier,
entrapment and immobilization without carrier.
Adsorption, adhesion and covalent bound are type of immobilization on carrier, mainly porous
carrier. In the case of porous carriers, cells first migrate into the porous matrix before being fixed
to the carrier.
The entrapment method of immobilization is based on the localization of an microorganism
within the lattice of a polymer matrix or membrane. It is done in such a way as to retain
microorganism while allowing penetration of substrate. It can be classified into inclusion,
encapsulation and membrane. Encapsulation is used in a number of different industries with a
wide variety of techniques or processes available.
Immobilization without carrier, such as cross-linking and flocculation based on covalently
bonded to a matrix through a chemical reaction or natural properties to agglomeration.
All methods of immobilization has advantages and drawbacks. The choice ofthe appropriate
methoddepends on the function of the application, type of microorganism, technical and
economic criteria.
One of the most important criteria is the cost of immobilization in food production, medical or
pharmaceutical applications. Microorganism immobilization has a broad range of application.
124
NANOMATERIALS FOR BIOANALYSIS AND BIOTECHNOLOGY
APPLICATIONS.
Dorota Szczepańska
Faculty of Chemistry, Gdańsk University of Technology, Gabriela Narutowicza 11/12, 80-233 Gdańsk, Poland
Over the last two decades, researchers have been working on the advancement of new
biotechnology applications of innovative nanomaterials. Nanomaterials possess unique size- and
material-dependent properties which make them attractive for improving regular biomedical
fields, such as drug delivery, imaging, therapy, and diagnostics.Applications of nanomaterials in
the biotechnological field are highly complex. A wide variety of nanoparticles shows potential for
these applications. Some of these nanomaterials have a drastic difference in their potentiality,
characterization and synthesis.
125
MYSTERIOUS CYTOTOXICITY ASSESSMENT OF ZINC OXIDE
NANOPARTICLES IN CELL LINES HELA, NIH-3T3 AND A-549
Szczepańska Izabela1
1
Warsaw University of Life Sciences
Zinc oxide nanoparticles (nZnO) are increasingly being used in products intended for humans or
animals and their safety of utilization is still unexplained. In this study the cytotoxic effect of
nZnO have been examined in cell lines such as HeLa, NIH-3T3, and A-549, which represent the
various target tissues exposed for nZnO. It has been shown that nZnO in concentrations from 10
to 200 μg/ml induces cytotoxic effect directly proportional to dose and duration time (30 - 240
min.), and this effect is different in the examined cell lines. Cell line A-549 was the most
sensitive to the toxic effects of nZnO, with reported value of IC50 of 24 μg/ml after 30 min.
incubation. HeLa is the most resistant line with value of IC50 of 145 μg/ml after 30 min
incubation, and 63 μg/ml after 240 min incubation. The results indicate that the evaluation of
nZnO toxicity should be conducted at the cells of target tissue representing interactions with
nZnO after application in humans or animals.
Bibliography:
[1] Lin WS, Xu Y, Huang CC, Ma YF, Shannon KB, Chen DR, Huang YW. 2009, Toxicity of nano- and micro-sized
ZnO particles in human lung epithelial cells. Journal of Nanoparticle Research, 11(1): 25-39
[2] Decksakulthorn F, Hayes A, Bakand S, Joeng L, Winder C. 2007, In vitro cytotoxicity assessment of selected
nanoparticles using human skin fibroblasts. AATEX, Special Issue: 397-400
[3] Guan, R., Kang, T., Lu, F., Zhang, Z., Shen, H., & Liu, M. 2012. Cytotoxicity, oxidative stress, and genotoxicity
in human hepatocyte and embryonic kidney cells exposed to ZnO nanoparticles. Nanoscale research letters, 7(1), 602
126
THE EFFECT OF SELECTED DIETARY SUPPLEMENTS ON THE
ACTIVITY OF GLYCOLYTIC ENZYMES
Justyna Szpotan1, Rafał Matusiak1 , mgr Iwona Majewska
1
Lodz University of Technology, Faculty of Biotechnology and Food Sciences, Institute of Technical Biochemistry,
Stefanowskiego 4/10, 90-924 Łódź, Poland Scientific Association of Biotechnology Students 'Ferment'
In 2010 according to the InternationalDiabetes Federation 6.4% ofthe world's population were
people suffering from diabetes. The rising amount of diabetics isrelated to changes inlifestyle
andincreased consumption ofhigh-carbohydrate foods. Antidiabetic drugs which are used now in
diabetes treatment are still insufficient and cause side effects. In traditional medicine white
mulberry (Morus alba) is used as an alternative to drugs. It is believed that the extract of leaves
can help to stabilized blood glucose level due to influence on the sugar digestion.
The reductionor slowing down thedigestion and absorptionof carbohydratesfrom thefood is one
of the possibilitiesofloweringpostprandialhyperglycemia. The starch digestiontakes place mainly
inthe intestineby the action ofthe enzyme called pancreatic α-amylase. Further, smaller fragments
of starch are hydrolyzed byα-glucosidase toabsorbablemonosaccharide - glucose. By
inhibitingenzymes involvedin the digestionof carbohydrates, glucose resultingfrom the
decomposition ofstarchgets into the bloodmore slowly. It is very important especially in a state of
insulin resistance in whichimpairedinsulin sensitivity results in glucose uptake from blood to
muscle only at a low level.
The aim ofour workwas to evaluate theability to inhibitα-amylase and α-glucosidase by white
mulberry (Morus alba) dietary supplements and extract of white mulberry leaves. Results of the
projectgave us an answerwhether tested mulberry dietarysupplements actually have
antihyperglycemic activity.
127
APPLICATION OF CONFOCAL MICROSCOPY IN PLANT RESEARCH
Dawid Szpott1, Mateusz Mazurkiewicz1, Marek Pasiński1, Iwona Jędrzejczyk2
1
Scientific Association of Biotechnology Students „Biox‟
2
Department of Plant Genetics, Physiology and Biotechnology University of Technology and Life Sciences,
Kaliskiego Ave. 7, 85-796 Bydgoszcz, Poland
The confocal microscopy has become ahelpful tool for a wide range of investigations in the
biological and medical sciences. The increasing availability of this equipment has begun a
revolution in plant biology in which microscopy has again become a powerful tool for
understanding structure and function of the organism.
This system offers several advantages including the ability to control depth of field, elimination
or reduction of background, and the capability to collect serial optical sections even from thick
specimens. The use of multiply-labeled specimens, different probes can simultaneously identify
several target molecules simultaneously, both in fixed specimens and living cells and tissues. The
confocal microscopy have made possible multi-dimensional views that include image
information in the x, y, and zdimensions as a function of time. The confocal microscopy together
with modern staining techniques enables studies on cell organisation as well asfollowing
processes taking place within the cell or tissue.
In the present work we will focus on the application of this technique in the investigation of
different plant cell structures e.g. nuclei, mitochondria, cell wall and cytoskeleton.
128
Some doubts about the tetrazolium salts
Marcin Szustak
Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Łódź University of Technology,
Stefanowskiego 4/10, 90-924 Łódź, Poland, Scientific Association of Biotechnology Students FERMENT
The inconvenience arising from the use of cell viability assays based on the use of negatively
charged dyes or hazardous to health and requiring specialized equipment isotopic methods, have
forced scientists to develop a simpler and much safer method. It has become very popular to use
tetrazolium derivatives as viability tests‟ components what not only reduces risks connected with
the use of radioactive elements, but also significantly increases the efficiency of the
measurement. Developed in 1986 method applying (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), due to previous mentioned advantages combined with low
costs, quickly found a lot of followers. What is more, during the following years, a numbers of
modifications of MTT assay to rectify most of its inconvenience have been created. Although this
technique has been adapted in many laboratories around the world, more and more often reports
suggesting a significant limitations in the use of tetrazolium salts appear in the literature. It turns
out that final results of the experiment are influenced by many additional factors. To clearly
determine whether this assay is suitable for a particular experiment it is required to perform
preliminary comparative trials.
Conducted studies were focused on the MTT assay evaluation in the case of its usage for
analyzing the mouse insulinoma β-TC3 cell viability under the influence of selected colorful
plant extracts.This method is based on the formation of purple formazan crystals from yellow
tetrazolium salt. Obtained results were compared with commercially available Presto Blue™
assay based on the reduction of the fluorescent dye resazurine.
129
CHANGES IN DNA METHYLATION PROFILE IN MAIZE UNDER
HERBICIDE STRESS
Jakub Szymkowiak1, Joanna Gracz1, Tomasz Twardowski1, Agata Tyczewska1
1
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland
Maize, Zea mays, a plant that originally comes from South America is currently cultivated in the
world on a large scale. Its great potential is broadly used in food and fodder industry, textile
industry but also in bioethanol production. Maize cultivation area is constantly increasing, this
and the fact that weeds that grow in the corn field develop resistance to widely used herbicides
are main reasons why the dosages of these chemical compounds had to be increased.
Biological organisms constantly exposed to environmental stimuli named stresses are capable of
establishing mechanisms of protection and adaptation. Because of their sedentary life style, plants
are restricted to tolerance, resistance, and avoidance mechanisms only and thus require efficient
short-term strategies based on the manipulation of the existing genetic information. Recent
studies have indicated that regulation of stress-responsive genes often depends on chromatin
remodeling, which is governed by processes often associated with epigenetic regulation (DNA
methylation, histone variants, post-translational modifications). It has also been hypothesized that
stresses could reshape a genome on epigenetic level via transpose activation. The fraction of
maize genome that appears to be repetitive sums to 85%, and there is a significant change in
methylation level in plants showing differential resistance to herbicides.
It has been observed in the fields that some maize lines display higher resistance to herbicides
than others but to this day the molecular mechanisms of such resistance remain unknown. To
detect changes in DNA methylation under herbicide stress in two maize lines displaying different
susceptibility to RoundUp® we used MSAP (Methylation Sensitive Amplified Polymorphism)
method and we observed differences in methylation profiles between the two tested lines.
130
THE EFFECT OF OSMOTIC PRESSURE ON ERYTHRITOL
PRODUCTION FROM GLYCEROL BY YARROWIA LIPOLYTICA
Ludwika Tomaszewska1, Magdalena Rakicka1, Krzysztof Cybulski1
1
Katedra Biotechnologii i Mikrobiologii Żywności, Uniwersytet Przyrodniczy we Wrocławiu, ul. Chełmońskiego
37/41, 51-630 Wrocław
Erythritol is a natural sugar alcohol with the 60-80% of the sweetness of sucrose. Its application
in food production was recognized as safe and approved throughout much of the world. Erythritol
is desireable food additive because it has very low caloric value (0 – 0.2 kcal/g), similar
rheological properties to sucrose, lack of off-taste and odors and is well tolerated by gastric
system even when consumed with an excess. Among polyols that are used as sucrose
replacement, it is the only one that is commercially produced in biotechnology processes, as its
production by chemical methods is not effective. Although many yeast genus are known to
produce erythritol, higher yields have been obtained with the application of osmophilic
microorganisms.
The aim of this study was to examine the impact of osmotic pressure, caused by the addition of
sodium chloride, on erythritol production from glycerol by Yarrowia lipolytica Wratislavia K1.
The erythritol biosynthesis was carried out in the shake-flask cultures in glycerol media (100
g/L). The applied NaCl concentration in the media was in the range of 0 – 14%, added in whole
at the beginning of the culture or divided into two portions (the first added at the beginning and
the second after 24 h of the cultivation).
In the cultures the amount of produced erythritol depended on NaCl concentration, as well as the
method of its addition. The addition of the salt enabled to increase the concentration of erythritol
and its purity, respectively from 19.4 g/L and 54% in the control up to 27.2 g/L and 91% when
4% of NaCl was added by two portions (20+20 g/L). In such conditions erythritol production
yield, volumetric and specific production rates were the highest and reached 0.32 g/g, 0.16 g/Lh
and 0.021 g/gh, respectively. Moreover, high erythritol production yield were observed when
initial osmotic pressure of the media ranged 2.7 – 4.0 Osm/kg. In turn, erythritol productivity was
found to be the highest when osmotic pressure was maintained at the level of 2.7 – 3.4 Osm/kg
throughout the process.
This work was co-financed by the European Union as part of the European Social Fund.
131
ENZYMATIC HYDROLISIS OF DEXTRAN PRIOR LC-MS/MS
DETECTION IN URINE SAMPLES FOR ANTI-DOPING CONTROL
Kamil Filip Trzebuniak1, Piotr Chołbiński2, Dorota Kwiatkowska2, Maria Balcerzak1, Andrzej
Pokrywka2
1
Politechnika Warszawska, Wydział Chemiczny, Katedra Chemii Analitycznej, ul. Noakowskiego 3, 00-664
Warszawa
2
Instytut Sportu, Zakład Badań Antydopingowych, ul. Trylogii 2/16, 01-982 Warszawa trzebuniak@gmail.com
Dextran (DEX)is a branched polymer of glucose withα-1,6‟-O-glycosidic bonds, produced by
some lactic-acid bacteria. Thissubstanceis widely used in medicine as blood plasma volume
expander, especially in case of hypovolemia. It may also be used by athletes mainly to mask the
use of other doping agents or to change the haematocrit value. There are also reports of dextran
misuse in endurance sports (e.g. marathon) to prevent dehydration.
The aim of this study was to develop a detection method fordextran screeningby using high
performance liquid chromatography coupled with tandem quadrupole-time-of-flight mass
spectrometry (HPLC-ESI-QTOF-MS). The method is based on the detection of isomaltose from
enzymatic DEX hydrolysis. It was validated according to World Anti-Doping Agency technical
documentation.
The study was performed at the Department of Anti-Doping Research Institute of Sport in
Warsaw (pol. Zakład Badań Antydopingowych Instytutu Sportu w Warszawie).
132
Cellulose as a new polymer for the immobilization by extrusion method
Dawid Urbański1, Magdalena Stobińska1
1
Center of Bioimmobilisation and Innovative Packaging Materials, West Pomeranian University of Technology,
Szczecin, durbanski@zut.edu.pl
Cellulose is the most common biopolymer on Earth, which is produced by plants and bacteria. Its
content depends on biological origin (wood – 40-50%, cotton fiber – 90%, becterial cellulose –
up to 100%). Cellulose consists the D-(+)-glucose units linked β(1-4)-glycosidic bonds and
creates linear and unbranchedmacromolecular chain with high molar mass. This structure allows
for tightly packing of polymeric chains linked by hydrogen bonds, which gave the cellulose high
stiffness and mechanical strength. Despite good mechanical properties it remains very sensitive to
biological degradation and hydrolysis especially in high concentrated acid conditions.
Even though cellulose itself has a high polarity, due to numerous hydroxyl groups it is insoluble
in water and in most organic solvents. For this reason there is a necessity to apply so called next
generation solutions or solvents, for example Schweizer reagent, ionic liquids (ILs) or Nmethylmorpholine N-oxide (NMMO). However, to dissolve cellulose fibers, these solvents
require high temperatures approximately 100°C and high concentration of environmentally
unacceptable chemicals. In recent years, it was developed a new solvent which is based on
aqueous solution of NaOH and urea mixture. After addition the cellulose to such solution, it is
formed a milky colloidal suspension., which by cooling at specific temperature leads to
transformation to transparent solution. Recently it was confirmed that this kind of solution allows
to generate a hydrogel cellulose beads by precipitation method at room temperature.
The aim of this study was to determine porosity cutoff and crushing strength of gel
beads.Porosity cutoff was determined by using dextran molecules with different molar masses
and by HPLC technique. The strength tests were carried out on a testing machine Zwick Roell
Z/2.5. The results of these studies will allow to continue the further development of the novel
method to generate cellulose hydrogel capsules with liquid core/membrane structure by coextrusion method.
133
THE INFLUENCE OF NRF2 TRANSCRIPTION FACTOR AND HEME
OXYGENASE-1 ON OSTEOCLASTOGENESIS
Monika Viscardi, Urszula Florczyk, Alicja Józkowicz, Józef Dulak
University, Kraków, Poland
Introduction: Osteoclastogenesis is a process of bone-resorbing osteoclasts (OCL) formation
from monocyte-macrophage precursor cells.Increased number and activity of OCLs are
associated with intense bone demineralization in disorders such as arthritis or osteoporosis.
Recent studies suggested the involvement of the cytoprotective Nrf2 transcription factor and its
target, heme oxygenase-1 (HO-1) in osteoclastogenesis, however, detailed mechanism still need
to be examined.
Aim: The aim of the study was to investigate the effect of Nrf2 and HO-1 deficiency in murine
bone marrow macrophages on osteoclasts differentiation.
Materials and methods: As a model we used Nrf2- or HO-1-deficient mice (Nrf2-/- or HO-1-/-,
respectively) and appropriate wild-type mice as a control (Nrf2 +/+ or HO-1 +/+, respectively).
Bone marrow cells were isolated from tibial and femoral bones and stimulated for 3 days with
macrophage colony-stimulating factor (M-CSF) to obtain bone marrow macrophages (BMMs)
and for the next 3 days with M-CSF and receptoractivator of nuclear factor kB ligand (RANKL)
for OCLs differentiation. At day 3 the level of BMM surface markers (F4/80, Mac-1) were
analyzed, while at day 6 the level of OCL markers were checked such as nuclear factor of
activated T-cells, cytoplasmic 1 (Nfatc-1), calcitonin receptor (Calcr), cathepsin K (Ctsk) and
integrin beta 3 (Itgb) using real-time RT PCR. At day 6 RANKL-stimulated BMMs were stained
also for tertrate-resistant acid phosphatase (TRAP), an enzyme specific for mature OCLs, and
violet multi-nucleated cells (≥ 3 nuclei) were counted as OCLs.
Results: The mRNA level of Mac-1 and F4/80 surface markers were increased in BMMs after
treatment with M-CSF irrespective of the genotype. However, using TRAP activity assay we
showed that Nrf2 deficiency resulted in higher number of OCLs , while less TRAP+ cells were
detected when HO-1-/- was absent, as compared to appropriate controls. In accordance with the
letters, osteoclasts-specific genes (Nfat-c1, Calcr, Ctsk, Itgb) were all upregulated in RANKLstimulated HO-1-/- BMMs in comparison to HO-1+/+ BMMs. On the other hand, an approach of
Nrf2 activation by sulforaphane (SFN) in RANKL-stimulated Nrf2+/+ BMMs confirmed that
Nrf2 has inhibitory effect on OCLs formation. After SFN treatment no TRAP+ cells
corresponding to OCLs were observed.
Conclusions: HO-1 deficiency in BMMs impairs osteoclasts formation, while the lack of Nrf2
enhances osteoclastogenesis.
134
FARMACOGENETICS OF ANESTHETIC AND ANALGESIC AGENTS
Michał Walczak1
1
Uniwersytet Przyrodniczy w Poznaniu
Pharmacogenetics is the study of the molecular mechanisms that underlie individual differences
in drug metabolism. Heritable changes in genes encoding drug metabolizing enzymes often
affects outcome in drug treatment. A knowledge of which drug and dosage would be most
effective for individual patient or might induce adverse reactions will significantly improve
medical care. Rapid advance in DNA sequencing and genotyping methods have enabled to
predict patients response for drug therapy and relate gene polymorphism with defined reactions.
It helps to select relevant drug treatment for individuals and also reduce adverse reactions.
Pharmacogenetics is a very useful tool in anesthesiology, where acute, postoperative pain and
undesirable side effects often appear as a consequence of wrong adopted drug dosage.
Polymorphic variations may significantly affect drug absorption, distribution, metabolism and
toxicity. It is important to remember that genetic underground may be not only factor involved in
drugs impact for individuals, we distinguish also environmental factors. Therefore it could be a
complex phenomenon. Finally pharmacogenetics data may led to elaborate genetic screening test
with effective application in drug therapy.
135
HISTONE DEACETYLASE INHIBITORS AS A NEW
MULTIFUNCTIONAL ANTICANCER DRUGS
Anna Wawruszak1,2 , Karolina Okła2, Monika Orzełowska2, Joanna Haliniak2, Joanna Florczak2,
Paulina Cieśla2, Artur Anisiewicz2
1
Katedra i Zakład Biochemii i Biologii Molekularnej Uniwersytetu Medycznego w Lublinie
2
Studenckie Koło Naukowe „Mikron”; Uniwersytet Marii Curie-Skłodowskiej w Lublinie
Acetylation and deacetylation of nucleosomal histones play an important role in the modulation of
chromatin structure, chromatin function and in the regulation of gene expression [1].Histone
deacetylase inhibitors (HDACi) are a promising new class of chemotherapeutic drugs currently in
early phase clinical trials. HDACs are enzymes involved in the remodelling of chromatin, and have
a key role in the epigenetic regulation of gene expression [2].HDAC and histone acetyltransferases
exert opposing enzymatic activities that modulate the degree of acetylation of histones and other
intracellular molecular targets, thereby regulating gene expression, cellular differentiation, and
survival [3]. Aberrant transcription due to altered expression or mutation of genes that encode
HATs, HDACs or their binding partners, is a key event in the onset and progression of cancer. The
remarkable tumour specificity of these compounds, and their potency in vitro and in vivo,
underscore the potential of HDAC inhibitors as exciting new agents for the treatment of cancer [4].
In recent years, inhibition of HDACs has emerged as a potential strategy to reverse aberrant
epigenetic changes associated with cancer, and several classes of HDAC inhibitors have been found
to have potent and specific anticancer activities in preclinical studies [5].
Fig.1. http://www.ifom-ieo-campus.it/research/chiocca.php
References
[1] S.Mei, A.D. Ho, U.Mahklnecht; Role of histone deacetylase inhibitors in the treatment of cancer; Journal of
Oncology; 2004.
[2] R.Lindemann, B.Gabrielli, R.Johnstone; Histone-Deacetylase Inhibitors for the Treatment of Cancer; CellCycle; 2004.
[3] C. Midsiades; Novel Histone Deacetylase Inhibitors in the Treatment of Thyroid Cancer; Cancer Therapy: Preclinical;
2005
[4] R. Johnstone; Histone-deacetylase inhibitors: novel drug for the treatment of cancer; Nature Reviews Drug Discovery,
2002
[5] J.Bolden, M. Peart, R. Johnstone; Anticancer activities of histone deacetylase inhibitors; Nature Reviews Drug
Discovery; 2006
136
MODIFICATION OF POLYSULFONE Ag/SiO2
Renata Wawrzaszek1, Urszula Wawrzaszek2
1
Department of Experimental Surgery and Biomaterials Research Medical University in Wroclaw
2
Division of Engineering Surface, Catalysis and Corrosion.Faculty of Chemistry Wroclaw University
of Technology
Medicine begins to benefit from solutions offered by nanotechnology, using techniques and
structure creation methods in which at least one of the projected dimensions does not exceed 100
nm. The use of nanotechnology in medicine may prove profitable, helping to solve many of
problems hitherto existing and being dealt with in many of research laboratories worldwide. It is
well to remember, however, that novelties in the field inspired by this rapidly developing field of
technology should not only provide beneficial solutions but also be free of threats. Components
used in the process of synthesis must be biocompatible, should not cause allergy, initiate
inflammation, nor have carcinogenic or toxic effects.
The material should possess resilience to biological environment factors and be physically and
chemically neutral (create strong bindings with wet tissue and posses the capability of
intensifying the process of creating blood clot, wound healing and tissue regeneration) minimizes
discomfort for their patients, yet produces a good cosmetic outcome.
Preparation of polysulfone samples with Ag/SiO2 added. The test material is durable and
maintains a very stable in Ringer solution.The results of mechanical tests do not result in
significant changes in mechanical properties, analysis DSC showed the addition of nanofillers not
cause a significant change in the structure of the polymer. These polymers are rigid, high-strength
it has very high dimensional stability. Its glass transition temperature is 183- 185 °C.
137
MICROBIAL CORROSION OF METALS
Urszula Wawrzaszek1, Renata Wawrzaszek2,
1
Division of Engineering Surface, Catalysis and Corrosion, Wroclaw University of Technology,
2
Department of Experimental Surgery and Biomaterials Research Medical University in Wroclaw
Microbial corrosion is caused by corrosion and the presence of live microorganisms include
bacteria, fungi or algae on the surface of the metal. The presence of microorganisms contributes
to the formation on the metal surface so-calledbiofilm. Biofilm is a product of complex
multicellular organisms with one or more species or genera. Colonization is initiated within an
hour and becomes well established in the period of several days to several weeks, depending on
local conditions. The microorganisms can grow under aerobic and anaerobic conditions in an
aqueous medium in the soil or air. Most developing water or aqueous solutions containing
organic compounds and inorganic compounds. The resulting micro-organisms rapidly adapt to
environmental conditions, regardless of the pH (from 0 to 13), temperature (from -5 C to 120 
C), salinity or impurities present.
Theprocess ofcorrosion of metalsusuallycontribute: sulfate-reducing bacteria, sulfur-oxidizing
bacteria and sulfidesand bacteria: nitrogen, oxygen, manganese, ironand microorganisms
fermentorganic compounds.
Microorganisms constitute a serious threat to metallic materials. It contributes to the creation or
increase aggressiveness corrosive environments, the growth rate of anodic and cathodic
processes, damage and weakening of the material for example by reducing the tensile strength, as
well as contribute to crushing and delamination of coatings used to protect the metal.
References:
B. Kołwzan, Ochrona środowiska, Nr 4, 2011
J. Baszkiewicz, M. Kamiński, Korozja materiałów, Warszawa 2006
S. Kakooei, M. Che Ismail,B. Ariwahjoedi, World Applied Sciences Journal 17 (4), 2012
www.efcweb.org/efcweb_media/Downloads/EFC+WP10/MICbook.pdf, 04.11.2013
138
FRUITS OF SELECTED SPECIES OF FOREST PLANTS AS A SOURCE
OF HEALTHY ANTIOXIDANT SUBSTANCES IN THE DIET OF BIRDS
Adrian Witczak1*,Agata Nowogórska2, Monika Skwarek2, Jacek Patykowski2
1
Institute of Forest Sciences, University of Łódź, Branch in Tomaszów Mazowiecki, 97-200 Tomaszów Mazowiecki,
Konstytucji 3 Maja 65/67, Poland, *adrian.witczak@uni.lodz.pl
2
Łódź, 90–237 Łódź, Banacha 12/16, Poland
Forest fruits are one of the main food source for birds during winter. They provide not only the
energy substances, but also phenolic compounds which additional health promoting properties.
Free radicals and reactive oxygen species play a key role in the pathogenesis of many diseases.
Numerous studies have shown that secondary metabolites derived from plants can act as
antioxidants and support the natural defense against free radicals. Forest fruits have high
antioxidant activity mainly due to the high content of tannins, catechins, flavonoids, isoflavones
and phenolic acids.
The purpose of this study was to determine the antioxidant properties of the extracts from trees
and forest shrubs, and to analyze the impact of the content of total phenolic compounds on the
antioxidant properties of the studied materials. The contents of phenolic acids and flavonoids
were also determined. Fruits of sea buckthorn (Hippophaë rhamnoides), common hawthorn
(Crataegus monogyna), midland hawthorn (Crataegus laevigata), rowan (Sorbus aucuparia),
viburnum reef (Viburnum opulus) and black cherry (Prunus serotina) were used for the
experiments.
We showed that the highest flavonoid content was found in black cherry fruit and the smallest in
viburnum reef while the highest content of phenolic acids was found in the fruits of midland
hawthorn. The highest content of total phenolic compounds in the analyzed extracts was found in
midland hawthorn fruit and a similar level was determined in common hawthorn fruit, while the
smallest content was observed in rowan fruit. There was a positive correlation between the total
phenolic content and the antioxidant activity of the tested fruits.
139
LITTLE DOMESTIC KILLERS
Natalia Witerska, Paweł Rusinek
Uniwersytet Marii Curie-Skłodowskiej w Lublinie
The so-called microorganisms, that is organisms predominantly microscopic, are present in every
single part of the biosphere. We do not even realise how many potentially dangerous
microorganisms live around us. Some of them are simply neutral or even helpful but become
hostile under specific conditions. The purpose of our work is to analyze the negative influence of
these little beings. We will take into account the microorganisms‟ natural enviroment, the way
human organisms become infected and the conditions under which the infection is possible, as
well as the diseases caused by these infections. We will also answer the question of how (and if)
people should guard themselves against pathogens. We will rate the efficiency of commonly used
disinfection methods and compare selected species of bacteria and fungi (e. g. Clostridium
botulicum, Salmonella, E. coli, Listeria monocytogenes), analyze the influence of aforementioned
taxa groups on the development of allergy.
140
THE EFFECTS OF THE EXOGENOUS CHOLESTEROL ON
MYOFIBROBLASTIC TRANSITIONS HUMAN BRONCHIAL
FIBROBLASTS DERIVED FROM ASTHMATIC PATIENTS
D. Wnuk1, M. Kosińska1, J. Czyż1, M. Michalik1
1
Jagiellonian University, Faculty of Biophysics, Biochemistry and Biotechnology Department of Cell Biology,
Gronostajowa 7, 30-378 Kraków, Poland
The complexity of asthma pathogenesis is illustrated by two processes: chronic inflammation and
bronchial wall remodeling in the respiratory tract resulting from local overproduction of cytokines
and growth factors, mainly TGF-β1. Prolonged exposure of bronchial fibroblasts to TGF-β1 drive to
overproduction of extracellular matrix components (ECM) and phenotypic shifts result in
subepithelial fibrosis. Myofibroblasts are characterized by the presence of α-actin positive stress
fibers typical for smooth muscle cells and physiological hallmarks of fibroblasts [1].
Multiple studies in the Department of Cell Biology conducted in cooperation withDepartment of
Medicine atJagiellonian University Medical School have demonstrated the increased propensity
of “asthmatic” bronchial fibroblasts (HBFs) to myofibroblastic transition (FMT), in comparison
to fibroblasts derived from non-asthmatic patients [2]. However, the mechanisms underlying
these phenotypic shifts still remain unclear. We demonstrated that inhibitors of cholesterol
biosynthesis pathway (statins and zaragozic acid A) attenuated TGF-β1-induced FMT in
“asthmatic” HBFs. Inhibition of cholesterol synthesis leads to the suppression of the nuclear
translocation of p-Smad2 proteins. It indicates that, the intensity of TGF-β1-induced FMT in
HBFs is highly dependent on the intracellular cholesterol levels [3].
The intracellular cholesterol level can be dependent on the activity of biosynthetic pathway and
LDL-endocytosis, consequently we examined the effects of the exogenous cholesterol on the
intensity of TGF-β1-induced FMT in HBFs derived from asthmatic patients. Our preliminary data
suggest that phenotypic shifts undergone by “asthmatic” HBFs induced by TGF-β1 are promoted
under reduced extracellular cholesterol level, independently of Smad2 signaling pathway.
References:
[1] Makinde T, Murphy RF, Agrawal DK. The regulatory role of TGF-β in airway remodeling in
asthma. Immunology and Cell Biology. 2007;85(5):348–356.
[2] Michalik et al., Asthmatic bronchial fibroblasts demonstrate enhanced potential to
differentiate into myofibroblasts in culture. Med Sci Monit, 2009, 15, BR194-BR201.
[3] Michalik et al., Lovastatin-induced decrease of intracellular cholesterol level attenuates
fibroblast-to-myofibroblast transition in bronchial fibroblasts derived from asthmatic patients.
Europ J Pharmacol, 2013, 704 (2013) 23–32
141
MOLECULAR MIMIKRY IN AUTOIMMUNE DISEASES
Agnieszka Wojtoń1, Iwona Konieczna2
1
Biotechnology Academic Circle-Mikroby; Departament of Microbiology, Jan Kochanowski
University, Kielce, Poland
2
Departament of Enviroment Protection and Modeling, Jan Kochanowski University, Kielce,
Poland
Introduction. Balance in immune system is essential for keeping appropriate function of human
organism. The main aim of immune system is elimination of pathogens and keepingof specific
level of tolerance towards to self and foreign antigens. Molecular mimicry is common strategy
adopted by infections agents. We could define this phenomenon as similarity of structure or/and
sequence between microbial and host molecules. Mimicry between foreign and self molecules
may lead to cross-reactions and finally cause development of autoimmune diseases.
Aim. Analysis of described in literature events of molecular mimicry in autoimmune diseases
using bioinformatics methods.
Methods. Structures of bacterial and human proteins were obtained from NCBI or ExPASy
databases. For protein structure visualization a PDB Swiss Viewer was used. RasWin Molecular
Graphics was applied in structures superposition.
Results. Molecular mimicry in several autoimmune diseases was investigated. In analysis,
similarities between bacterial as well as viral proteins and human molecules was included. In few
cases, like described sequence similarities between human DNA topoisomerase and bacterial
proteins in systemic sclerosis, mimicry was not confirmed in structure analysis of abovementioned molecules.
Conclusions. Bioinformatic methods allow for detailed analysis of molecular mimicry essential in
autoimmune diseases.
142
HUMAN BREEDERS
Aleksandra Woźniak1, Dominik Salachna1
1
Imagine the world where everybody's beautiful , smart , strong , intelligent... just perfect.
Imagine that not everyone is supposed to live... That you can't make any decision in your life
because someone has already decided about your future before your birth... Just like you've been
"produced" in a some kind of "human factory" where "human breeder's" have the power to decide
who may live or not. Everything in the name of science and building better world. It's not a s-f
story or horror. It was started in 1883 when Francis Galton, a cousin of Carol Darwin named a
new genetic science - Eugenic.
There isn't such thing as "good" science or "evil" science. It's only a tool in humans hands. Let's
look together from XIX century in America through Adolf Hitlers visions to nowadays and try to
decide what's your opinion about this difficult and controversial issue.
Fig. Shot action from film "Eugenic: In the name of progress"
143
OBTAINING OF MUTANTS OF S. CEREVISIAE AND P. STIPITIS
KARYODUCTANT TO INTENSIFY THE PRODUCTION OF ETHANOL
FROM PENTOSES
Monika Wójcik1, Monika Kordowska-Wiater1
1
University of Life Sciences in Lublin, Department of Biotechnology, Human Nutrition and Science of Food
Commodities
A major problem in the production of bioethanol from ligninocellulosic biomass is the selection
of a suitable microorganism capable of simultaneous utilization of all the sugars contained in the
substrate (hexoses, pentoses) in high yield, which can tolerate high concentrations of
ethanol.Typical (known) S. cerevisiae strains used in the alcoholic fermentation are not capable of
fermentation of D-xylose, in turn, xylose-fermenting yeast P. stipitis are not resistant to high
concentrations of ethanol.Therefore, different techniques of genetic modification are used to
improve the ability of yeast to carry out the production of bioethanol, one of them is
karyoduction.
In these studies karyoductantS. cerevisiaeV30 and P. stipitis CCY39501 (SPK-8) was used. The
microorganism was subjected to UV-induced mutagenesis and the resulting mutants were tested
for tolerance to high concentrations of ethanol, and the efficiency of ethanol production from Dxylose.
The mutants were characterized by greater tolerance to ethanol as compared to the control strains
of Pichia stipitis and karyoductant SP-K 8, which were unable to grow on media containing 8%
ethanol. The mutant no. 112, 100, 98 showed a resistance to 8%, 10%, 12% (v/v) ethanol
concentration, respectively. In addition, five mutants were tested in terms of output of ethanol
production from D-xylose.Examined mutants showed significantly improved production of
ethanol: mutant No. 112 produced 0.48% (w/v) of ethanol in a yield of 0.29 g/g, mutant no. 110
produced a 0.52% (w/v) ethanol yield 0.28 g/g, mutant no. 100 and 106 produced 0.39% of
ethanol in a yield of 0.22 g/g, compared to the starting strain karyoductantSP-K 8 that produced
0.27% (w/v) of ethanol in a yield of 0.16 g/g. In breeding the mutant no. 98 the highest
production yield rate of 0.36 g/g was obtained at a concentration of ethanol of at 0.55% (w/v).
144
The importance of biofilms in fungal infections caused by Candida albicans.
Treatment and diagnosis of candidiasis
Justyna Wójtowicz1, Katarzyna Zarosa1
1
SKN Biotechnologów Mikron, Uniwersytet Marii Curie-Skłodowskiej, SKN Mikrobiologów Bakcyl, Uniwersytet
Marii Curie-Skłodowskiej
Candida albicans, a fungal pathogen, exists in different morphotypes. As commensals they
live in the digestive tract and on mucous membranes in the form of round blastoconidia. When
the host has very weakened immunity, Candida converts to the mycelium which consist of true
hyphae and pseudohyphae. In this form, fungi outgrow the tissue and cause fungal infections.
Candida albicans is one of the most common causes of hospital-acquired infections. It is able to
form biofilm on polymers from which are made for example heart valves, dental prosthesis,
catheters. The early diagnosis, correct pathogen identification and determination of their
susceptibility to the drugs are very important factors which are decisive in introducing a proper
therapy.
Amphotericin B is one of the oldest but also one of the most effective antimycotic drug.
Unfortunately, its usefulness is limited by toxicity, especially to the kidneys. The toxicity of AmB
is the reason why many scientists are looking for a new form of this drug with reduced the
toxicity to human cells and increased toxicity to the fungi.
145
ANTIBACTERIAL EFFECT OF THE CO-ADMINISTRATION OF
DENDRIMERS AND ANTIBIOTICS
Wrońska N.1, Zawadzka K.1, Lisowska K.1
1
The common use of antibiotics causes a rise in the resistance among disease-causing
microorganisms. This public health problem has increased especially for the last decade. Due to
this fact, alternative methods of bacterial infections treatment are being searched for. The
potential solution seems to be dendrimers - macromolecules with a three-dimensional structure
and well-defined physical and chemical properties.Because of their specific features they find
variety of application including biomedical uses such as the pharmaceutical delivery, gene
transfection, imaging agent. Most studies concerning the uses of dendrimers as drug delivery
systems. Literature data show that the binding of drugs with dendrimers improves their solubility,
stabilizes them in blood and prolongs their release. In recent years a few studies have also
documented that dendrimers can be used as potent antimicrobial drugs against Gram-negative
and Gram-positive bacteria.
From the literature data it is know that PEGylated PPI dendrimers loaded with ciprofloxacin
showed high antimicrobial activity against S. aureus and C. pneumonia.
The similar results were obtained with azithromycin conjugates of PAMAM dendrimers
Additionally, PAMAM dendrimers not only improve the solubility of nadifloxacin and
prulifloksacin but also enhance their antimicrobial properties.
Due to the fact, that common use of antibiotic also spread of resistant among microorganisms and
has contributed increase an environmental pollution, these research are very important.
Literature:
1. B. Devarakonda, R. A. Hill, W. Liebenberg, M. Brits, M. M. de Villiers,Int. J. Pharm.,
2005, 304, 193.
2. S. Kannan, P. Kolhe, V. Raykova, M. Glibatec, R. M. Kannan, M. Lieh-Lai, D. Bassett,J.
Biomater. Sci. Polym. Ed. 2004, 15, 311.
3. N. Malik, E. G. Evagorou, R. Duncan,Anticancer Drugs, 1999, 10, 767.
4. M. K. Calabretta, A. Kumar, A. M. McDermott, C. Cai,Biomacromolecules, 2007, 8,
1807.
5. K. Ciepluch, N. Katir, A. El Kadib, A. Felczak, K. Zawadzka, M. Weber, B. Klajnert, K.
Lisowska, A. M. Caminade, M. Bousmina, M. Bryszewska, J. P. Majoral,Mol
Pharm.2012, 9, 448.
6. Felczak , N. Wrońska, A. Janaszewska, B. Klajnert, M. Bryszewska, D. Appelhans, B.
Voit, S. Różalska, K. Lisowska, New J Chem. 2012, 36, 2215.
146
MUSHROOMS ARE NOT SO BLACK AS THEY ARE PAINTED
Adrian Zając1, Artur Pachla1, Michał Chojnacki1, Mateusz Pięt1
1
Scientific Students Group of Biotechnology „Mikron‟, Faculty of Biology and Biotechnology, Maria CurieSkłodowska University, adr25@poczta.onet.pl
Mushrooms are usually regarded as deprived of nutrition and healthysubstances, used only as
addition to food products in order toincrease their taste and aroma. Many people, also medicine
scientists, believe that mushrooms have no considerable influence on human organism if they are
included in diet and even as harmful and unnecessary. Recently it turned out that some species of
edible mushrooms are not as worthless as had been believed so far.
Many researches on pericarp of the mushroom are demonstrating that mushrooms are an
abounding source of biologically active compounds and also are valuable from point of dietary
view. The authors would like to present some selected species. Below are examples of
mushrooms to be presented:
Pleurotus ostreatus is a tasty edible mushroom, widely cultivated around the world, mostly in
Asia and Europe. Because of its capability to lignin degradation it can grow on dead trees and
agricultural wastes. It is a rich source of vitamins, proteins (especially high dose of essential
amino acids) and minerals with high potassium to sodium ratio, which in combination with high
content of lovastatin becomes good dietary compound for people suffering from hypertension and
heart diseases.
Lentinula edodes also called „Shiitake‟ is an edible mushroom from east Asia where is used in
cuisines and traditional medicine. It shows antiviral, antibacterial, anticancer and immune
modulating activity which makes it very valuable addition to the diet. The active compound of
Shitake, AHCC (α-glucan-rich compound), is sometimes used as alternative medicine in cancer
treatment.
Flammulina velutipes commonly called „Enoki mushrooms‟ are edible slender white mushrooms
with mild taste. They can be added to the dishes in raw form or after only brief cooking. It has
been shown that those mushrooms have significant anticancer and immune-enhancing effects
combined with high contain of antioxidants.
147
FILOVIRIDAE HAEMORRHAGIC FEVER VIRUSES
Olga Zaleska1, Olga Rodzik2
1,2
SKN Biotechnologów „Mikron”, Wydział Biologii i Biotechnologii, Uniwersytet Marii Curie- Skłodowskiej Lublin
Filoviridae are classified as hemorrhagic fever viruses that include pathogens of different viral
etiology. They are a group of several types of diseases with similar clinical pathology, the socalled bleeding diathesis. It is a symptom of combining these disease entities. They are founding
in specific regions of the world. However, development of air transport made possible for the
disease to be transmitted to areas where it does not naturally occur. Viral hemorrhagic fevers are
zoonoses. A reservoir of viruses responsible for hemorrhagic fevers are often animals. From
animals, these diseases can be transmitted to humans.
Filoviruses are among the most dangerous pathogen in the world. According to the recent studies,
a natural reservoir of the virus is considered to be bats, but the most common transmission of the
virus is through human contact with an infected animal or contact with the meat. The
development of communication is accompanied byan increased risk of introducing the pathogen
to the developed countries. The pathogen is also an object of desire for the production of
biological weapons. Because of these factors, the need for studying the mechanisms of
pathogenesis of these virusesincreases.
Today, many institutions in the world are working with Filoviroidae BSL 4 laboratories. Previous
studies have greatly expanded the knowledge on immune processes, although there still
existmany unresolved aspects. With these findings, it was possible to produce drugs and vaccines,
which are currently being tested mostly on animals.
References
Deptuła W., Niedźwiedzka P., 2008, Wybrane procesy immunologiczne związane z wirusami
gorączek krwotocznych., Uniwersytet Szczeciński, Szczecin, str. 523-524; 526
148
MICROBIAL DEGRADATION OF N-HETEROCYCLIC COMPOUNDS
Zawadzka Katarzyna1, Wrońska Natalia1, Bernat Przemysław1, Felczak Aleksandra1, Lisowska
Katarzyna1
1
N-heterocycles are polycyclic aromatic compounds that contain one or more N atoms in rings.
Pyridine, quinolone and carbazole have been detected in groundwater, soil and sediments. These
compounds are present in crude oil shale. Moreover, they are used in the production of
pharmaceuticals, dyes and insecticides. The presence of N-heterocyclic compounds in the
environment is particularly harmful because of their toxic and mutagenic character.
Microbial degradation of N-heterocycles can be an alternative to a more expensive physicochemical method of xenobiotics elimination from environment. In recentyears many researchers
have focused on the biodegradation of these pollutants. Data have shown that bacteria are able to
use N-heterocyclic compounds as a sole source of carbon, nitrogren and energy. However, little is
known about the degradation of N-heterocycles by microscopic filamentous fungi, which deserve
special attention due to the variety of metabolic pathways, low specificity of the enzymes
involved in the degradation process, high tolerance to high concentrations of toxic substances, as
well as the ability to spread in the soil by hyphal elongation, which facilitates the access to the
degraded compounds. The results of our previous studies indicate that the selected microscopic
filamentous fungi are capable of degrading N-heterocyclic compounds.Microorganisms used by
us comprise a pool of environmental strains, most of which were isolated from sites contaminated
with various xenobiotics.
Fig. 1. The chemical structures of pyridine, carbazole and quinoline
149
EMBRYONIC DIAPAUSE IN DOMESTIC SPECIES WITH SEASONAL
REPRODUCTION – TIME TO SOLVE THE ENIGMA
Kamila Zglejc
Scientific Association Biotechnologists, Faculty of Biology and Biotechnology, University of Warmia and Mazury in
Olsztyn, Tutor: dr hab. Iwona Bogacka
Embryonicdiapause(ED) is a conservativeevolutionary strategy, which has developed
independently indifferenttaxonomicgroups of mammals. Duringa delayedimplantation, the
fertilized egg, reversiblyarrestsown development inthe blastocyst stage, released from thezona
pellucida floating freelyin the uterus. EDoccurs in two forms: obligate–in all pregnancies and
facultative as a protective phenomenon that ensures optimal birth of offspring to survive
environmental conditions. The phenomenon ofdelayedimplantationoccurs inlessthan 2% of the
species of placental mammals (Artiodactyla, Rodentia, Insectivora, Carnivora, Chiroptera i
Edentata).
Studies have shown thatEDis also foundin domestic specieswithseasonalreproduction(sheep
andmare). Blastocysts entered into diapause, as demonstrated by growth arrest and their EDspecyfic pattern of gene expression. In diapausing ovine blastocysts, genes Proliferating Cell
Nuclear Antigen (PCNA) and signaling Heparin-binding EGF-like growth factor (HB-EGF) were
down-regulated. On the other hand anti-proliferative B-cell Translocation Gene 1 (BTG1) and
Cannabinoid Receptor Type 1 (CB1), which are normallydown-regulated before implantation,
were markedly up-regulated in diapausing ovine blastocysts.
The reproductive activity insheepand mares depends on affectedseason(environmental
conditions) and the lengthof day (melatonin secretion). In mares, breeding activity is generally
confined to the spring (longday). In the horse, which has an average gestation period of 336 days,
the incidence of extremely prolonged pregnancies (+30– 65 days) has been estimated to be ~1%.
In the horse it is possible to prove that a discrepancy between the excepted and actual length of
gestation is caused by delayed implantation.
Variousfactors (e.g. maternal stress)may contribute tothe occurrence ofdiapausein mammals
inwhichit is normally absent. It is assumed thatdiapausecan occur inallplacental mammals,
including humans.A restrictivedietlow inproteinmayalso contributeto the phenomenon
ofdelayedimplantation.EDmaybe a response to specific situations when further embryonic
development is imperilled due to e.g. harsh climates, temperature fluctuations or under-nutrition.
Embryonicdiapausestillremainsan evolutionaryenigmaof different speciesof placental mammals.
150
CROSS-LINKING APPROACH IN STRUCTURAL AND INTERACTION
STUDIES BETWEEN YIN-YANG 1 AND ITS MOLECULAR PARTNER
TATA-BOX BINDING PROTEIN
Dawid Żyła1,2, Katarzyna Wajda-Nikiel1,2, Anna Baranowska1,3, Andrzej Górecki1
1
Department of Physical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian
University, Kraków
2
Scientific Association of Biotechnology Students “Mygen”
3
Scientific Association of Biochemistry Students “N.zyme”
Covalent modificationsof proteins depend on availability of particular chemical compounds that
are capable of reacting with specific functional groups that are present in proteins. One of the
most important non-natural and covalent modification of proteins is cross-linking – the process of
chemical joining two or more molecules by a covalent bound. Reagents used in cross-linking
contain groups at the ends, which are reactive to specific functional groups (primary amines,
sulfhydryl groups, carboxyl groups, etc.). Cross-linking reaction might be used in interaction and
structural studies of proteins and their ligands.
The human transcription factor Yin-Yang 1 (YY1) regulates the transcription of remarkably many
genes and a number of its targets is still mount. YY1 exerts its effects on genes involved in these
processes via its ability to initiate, activate, or repress transcription depending upon the context in
which it binds. As an ubiquitously expressed transcription factor, YY1 has been demonstrated to
regulate cell proliferation, differentiation and may have potential regulatory function in different
types of cancers. YY1 is an intrinsically unstructured protein - the full length of YY1 has never
been crystallized for structure analysis, except for the cocrystal study of its zinc finger region
with the adeno-associated virus P5 promoter.
Our latest in vitro research indicates that YY1 interacts with human TATA box binding protein Cterminal domain (TBPc). TBP is a general transcription factor that binds specifically to a DNA
sequence called TATA box. TBP together with TBP-associated factors makes up part of RNA
polymerase II preinitiation complex. The role of the interaction with YY1 is unknown.
Using BS3 cross-linker, which is a homobifunctional (it has the same Sulfo-NHydroxysuccinimide reactive groups at both ends of spacer arm), water-soluble and aminereactive protein cross-linker, we want to define the interaction sites between YY1 and TBPc.
Cross-linked proteins will be digested with V8 protease and analysed using mass spectroscopy
for detection of cross-linked peptides. Finally we hope to see the difference between separately
cross-linked proteins and the complex. This research may bring a new insight for intrinsically
unstructured proteins studies, because methods like NMR and X-ray crystallography are not
eligible in that field of study.
151
THE ACTIVITY OF ETHANOL AND HEPTAN EXTRACTS OBTAINED
FROM CHAMOMILE (MATRICARIACHAMOMILLA L.) AGAINST
HUMAN COLON CANCER CELL LINE HT29 IN VITRO TESTS
Aleksandra Żurek1,2, Arkadiusz Czerwonka1, Maciej Frant1,2, Barbara Zdzisińska1, Grażyna
Matysik3
1
Department of Virology and Immunology, MariaCurie-SklodowskaUniversity,Lublin, Poland
2
Scientific Students Group of Biotechnology “Mikron”, MariaCurie-SklodowskaUniversity, Lublin, Poland
3
Department of Analytical Chemistry, MedicalUniversity, Lublin, Poland
Chamomile is a daisy-like plant that belongs to the family Asteraceae. It can be prepared as an
infusion which is well known from its beneficial effects, e.g. calming, anti-inflammatory and
soothing when used on mild skin irritations. Chamomile can be used as a therapeutic agent in
treatment of such afflictions as: common cold, cardiovascular conditions, eczema and
gastrointestinal conditions. Drinking chamomile tea is considered to boost the immune system
and to help overcoming infections such as cold, it also has calming effect on the patient‟s mood
so it‟s recommended in states of anxiety. Chamomile essential oil is widely used in aromatherapy
e.g. helping to improve the quality of life of patients with cancer and other serious health
problems. [1].
Scientific research shows that extracts obtained from various species of chamomile have some
anticancer activity, mainly against cervical and colon carcinoma [2, 3].
Our study aimed to examine and compare the cytotoxic effect of extracts obtained from
chamomile (ethanol and heptanic) against tumor cells from colorectal cancer (HT29 cell line).
For this purpose we conducted two tests: NR uptake and MTT assay. The concentrations of the
extracts were tested in the range of 25 μg/ml to 250 μg/ml.
The cytotoxic effect of both extracts was dose-dependent and varied, depending on the type of
extract. The heptanic extract showed strong cytotoxicity to the HT29 cell line in both tests
(decreased viability to 40% for NR uptake and to 5% for the MTT assay, at the highest
concentration of the extract). The ethanol extract also reduced the viability of HT29 cells
comparing to the control (reduction to 50% in MTT assay and 60-70% in NR uptake for the two
highest concentrations of the extract).
Srivastava J., Gupta S.: Chapter 8 – Health Promoting Benefits of Chamomile in the Elderly Population,
Complementary and Alternative Therapies and the Aging Population, 2009: 135-158
Guimarães R., Barros L., Dueñas M., Calhelha R. C., Carvalho A. M., Santos-Buelga C., Queiroz M. J. R. P., Isabel
Ferreira I.C.F.R.: Infusion and decoction of wild German chamomile: Bioactivity and characterization of organic
acids and phenolic compounds, Food Chemistry, 2013, 136 (2): 947-954
Guimarães R., Barros L., Dueñas M., Calhelha R. C., Carvalho A. M., Santos-Buelga C., Queiroz M. J. R. P., Isabel
Ferreira I.C.F.R.:Nutrients, phytochemicals and bioactivity of wild Roman chamomile: A comparison between the
herb and its preparations, Food Chemistry, 2013, 136 (2):718–725
152
WHAT IS HIDING IN ORGANIC CHEMISTRY LABORATORY?
Joanna Fedorowicz1
1
Politechnika Gdańska, Wydział Chemiczny, Katedra Mikrobiologii, Koło Studentów Biotechnologii
Students often do not respect the safety rules duringlaboratory classes. They get rid of the waste
from the experiment in a way not recommended. Organic substances, instead of getting into
specially dedicated for this purpose the waste cans, are poured into the sinks. Microorganisms
living in the sewer drains adapt to difficult condition. Continuous genetic variation can lead to
evolve new defense mechanisms against harmful chemical toxins. Bacteria and fungi may acquire
the ability to dispose chemical solvents and grow even in environments with high concentrations
of compounds that inhibit the growth. Resistance can propagate in the strain with cell division
and by the conjugation.
The subject of the study was to isolate microorganisms in the laboratory Department of Organic
Chemistry and tested them for resistance to organic chemicals commonly used in laboratory. The
project aim was to get strains of bacteria and fungal microorganisms capable of growing in the
presence of high concentrations of organic solvents.
In the laboratory room was taken some samples biological material by using swabs. Then they
was tested if there are any microorganisms (bacteria and fungi) by growth on media plates with
Sabourand and LB. To isolate pure strains was made a streaking. Strains was described and there
was made a bacterial lawn from them (LB plates for bacteria and Sabourand Agar for fungi).
Plates treated organic substances as ethanol, petroleum ether, chloroform, methanol, n-hexane
and isopropanol by soaking sterile rings of filter paper in a specific substance and laying them on
the surface of plates in appropriate distances. Therealso used 3 reference strains. Plates was been
observed.
In many cultures were obtained with a similar growth as reference strains, but succeeded in
discover some strains with more resistant to ethanol and highly growing in the presence of
petroleum ether and hexane.
Bacteria growing intensity around organic solvents are not only resistant to the tested
compounds, but also probably created mechanisms which enable them to use those toxic
substances as carbon source.
153
LIPOPOLISACCHARIDE STRUCTURE OF PATHOGENIC
ESCHERICHIA COLI STRAINS
Justyna Bazan1,2, Kinga Gostomska3, Katarzyna Dudziak2
1
Wroclaw Medical University, Department of Medical Biochemistry
50-368 Wroclaw, ul. Chalubińskiego 10 ,
2
Bartla 5,
3
Wroclaw University of Environmental And Life Sciences, 50-375 Wroclaw, ul. Norwida 25,
Most Escherichia colistrains are harmless, but some serotypes can cause serious gastrointestinal
bacterial infection in humans and animals. E. coli has been associated
with diarrhoea (particularly in children), haemorrhagic colitis, dysentery, bladder and kidney
infections, surgical wound infection, septicaemia, haemolyticuraemic syndrome, pneumonia and
meningitis. The mechanism of the diarrhea is associated with the crossing of the gastric barrier by
E. coli and proliferation of pathogenic strains producing enterotoxin in view
of the small intestine.
Lipopolysaccharide
(LPS,
endotoxin)
is
a
very
important
structure
of the Gram-negative bacteria cell wall. LPS consists of three components or domains:
Lipid A, core (R-polysaccharide) and an O-polysaccharide (O-antigen). O-antigen
is characteristic structure for smooth type LPS and in Escherichia coli it is responsible
for the pathological consequences of bacterial infection.
There are over 160 different O-antigen structures produced by Escherichia coli strains.
Appearance of so many different serological types is connected with huge diversity
in carbohydrates composition occurring within LPS molecule.
In this work we present the studies on identification and characterization of pathogenic strains of
E. coli isolated from infected animals. In particular, GLC-MS sugaranalysis
of O-antigens was performed.
References
Brzozowska E., et al Post Hig Med Dosw 2011; 65: 167-76
Bazan J. Sepsis 2011; 4(1): 95
Acknowledgments:
This work is co-financed by the European Union as part of the European Social Fund.
154
IDENTIFICATION OF PROBIOTICS BY DECONVOLUTED ATR-FTIR
SPECTRA
Barbara Kmiecik1, Anna Skotny2, Sylwia Olsztyńska-Janus3, Małgorzata Komorowska3
1
Wroclaw University of Technology, Institute of Materials Science and Applied Mechanics, ul. Smoluchowskiego 25,
50-370 Wroclaw, Poland
2
Wroclaw Medical University, Department of Internal Diseases, Geriatrics and Allergology, Wybrzeze L. Pasteura
4, 50-367 Wroclaw, Poland
3
Wroclaw University of Technology, Institute of Biomedical Engineering and Instrumentation, pl. Grunwaldzki 13,
50-372, Wroclaw, Poland
Probiotics recently become a very important part of the medicine. They have a positive effect on
condition of human body after antibiotic treatment. Many studies have shown that only a few
types of probiotics have a positive effect on our organism. Due to the fact that they are becoming
part of our diet, very important issue is information what kind of these microorganisms are in our
food. In these studies, identification of probiotics was made by using ATR-FTIR spectroscopy.
Researchers indicate region of 900-600 cm-1 as “fingerprint” area for these microorganisms [1].
However, our spectra show that the identification of probiotics can also be made by
deconvolution of amide I and II bands. Results of our studies confirm that this way also provide
to correct conclusion.
[1]Davis, R., and L. J. Mauer. "Fourier transform infrared (FT-IR) spectroscopy: a rapid tool for
detection and analysis of foodborne pathogenic bacteria." Current research, technology and
education topics in applied microbiology and microbial biotechnology. Formatex Research
Center, Spain (2010): 1582-1594.
155
GENETIC STRUCTURE AND FUNCTIONS OF THE CHROMOSOME OF
THE ARCTIC PSYCHROBACTER SP. STRAIN
Robert Lasek1, Łukasz Dziewit1, Dariusz Bartosik1
1
Department of Bacterial Genetics, Institute of Microbiology, Faculty of Biology, University of Warsaw;
Miecznikowa 1, 02-096 Warszawa; lasek@biol.uw.edu.pl
As a part of complex genomic studies of a pool of Arctic Psychrobacter spp. strains
(Gammaproteobacteria) we have recently determined the complete nucleotide sequence of the
genome of psychrophilic strain DAB_AL43B, which was isolated from ornithogenic deposits in
Spitsbergen (Svalbard Archipelago, Norway). The genome consists of one circular chromosome
(3.31 Mb) and 4 cryptic plasmids (4.4 – 6.4 kb).
We performed a detailed in silico analysis of the DAB_AL43B chromosome to elucidate its
structure and functions. A pool of genes encoding proteins engaged in the expression of the
genetic information was identified, including these involved in transcriptional regulation, the
response to the temperature shock, and DNA repair. We also distinguished mobile genetic
elements residing within the chromosome (transposable elements and a Mu-like prophage).
Interestingly, it is likely that some of these elements are responsible for large-scale chromosomal
rearrangements which have been detected within a part of bacterial population.
Based on the bioinformatic analyses, we reconstructed the network of basic metabolic pathways
of the DAB_AL43B strain, which comprised i.a. carbon, amino acid and energy metabolism. Our
findings were supported by the results of the metabolic analyses performed with the use of the
BIOLOG phenotype microarrays. We also identified a pool of genetic determinants which confer
adaptation to low temperatures. The comparative genomic analyses of Psychrobacter spp. were
also performed to establish the unique features of the DAB_AL43B genome.
Because of its metabolic characteristics and genetic potential, the analyzed strain of
Psychrobacter sp. is a good candidate to create host-plasmid systems that may prove useful in the
biotechnology of psychrophilic bacteria – a promising yet still understudied field.
156
EPSILON PROTEIN – AN ESSENTIAL ELEMENT IN PLASMID
pSM19035 STABILIZATION
Emilia Maciejewska
Warsaw University of Technology, Faculty of Chemistry
Plasmids are small genetic elements giving the bacterial cells possibility of survival in an
unfavorable environment. The toxin-antitoxin (TA) system is a strategy that protects bacterial
cells against the loss of the plasmid. An example of TA pair is Epsilon and Zeta - proteins
encoded on plasmid pSM19035 from Streptococcus pyogenes. This system is the type II of TA
systems which implies both components are proteins. Control of this system is based on the low
stability of antitoxin. When the daughter cell will not inherit the plasmid, antitoxin from TA
complex is degraded. As a result toxin is released which causes cell death. This mechanism is
called post segregational killing (PSK).
Research on proteases involved in Epsilon degradation process at in vivo conditions is impossible
because of low stability of the antitoxin. Thus, in a group led by Urszula Zielenkiewcz (Ph.D.) in
Department of Microbial Biochemistry, Institute of Biochemistry and Biophysics PAS, Clp and
Lon families of proteases from Bacillus subtilis are used in in vitro investigation of this
degradation process.
Purification of Epsilon and ClpP proteins and testing their properties, such as temperature
stability or salinity, were performed as a part of the engineering thesis. This allowed to prove that
these proteins, obtained by genetic engineering methods, are suitable to carry out the degradation
reaction in in vitro conditions.
157
VITAMIN D SIDE-CHAIN MODIFIED ANALOGS AND THEIR IMPACT
ON ACUTE MYELOID LEUKEMIA CELL LINES
Małgorzata Grudzień
Department of Protein Biotechnology, Faculty of Biotechnology, Uniwersytet Wrocławski
Vitamin D3 is a compound which is synthesized in skin and transferred to the liver and kidneys
where is transformed to its biologically active form – 1,25(OH)2D3. Vitamin D plays a role in
maintaining calcium and phosphate homeostasis in vertebrate organisms and is a ligand for
vitamin D receptor (VDR). VDR after binding the ligand, heterodimerizes with retinoid X
receptor (RXR) which increases VDR stability and promotes nuclear import.
VDR belongs to the nuclear receptors superfamily and containsseveral conservative domains.
DNA-binding domain (DBD) is responsible for recognizing target sequences in genes. Ligandbinding domain (LBD) is a region which mediates heterodimerization.
Vitamin D and its synthetic analogs are tested as a potential therapeutics in leukemia treatment.
Vitamin D is capable to stop proliferation in acute myeloid leukemia (AML) and to start
differentiation of the cells, unfortunately concentrations of vitamin D which ensure those
activitiescause hypercalcemia and hyperphosphatemia. Many vitamin D analogs were
synthesized and tested on AML cell lines, several exhibit appropriate properties for becoming a
drug in cancer treatment.
Experiments with analogs PRI-1906 and PRI-1907 showed that these compounds display high
cell – differentiating activity. The aim of my future study is to test new, side-chain modified
analogs of vitamin D2 (PRI-5100, PRI-5101, PRI-5201and PRI-5202) and their influence on
HL60 cell line differentiation and proliferation.
158
INVESTIGATION OF LYMPHATIC ENDOTHELIAL CELL DEATH
MECHANISM AFTER PDT
Agata Osiak1, Angelika Muchowicz1, Małgorzata Wachowska1
1
Department of Immunology, Centre of Biostructure Research, Medical University of Warsaw, Warsaw, Poland
Lymphatic vessels are involved in development of autoimmune diseases, inflammation and
tumors. Peritumoral lymphatics represent an integral part of tumor staging and contribute to
tumor metastasis. Therefore anti-lymphangiogenic therapy which can prevent tumor progression
is a such promising strategy.
Photodynamic therapy (PDT) is successful and minimally invasive therapeutic method used in
the treatment of various solid tumors and non-malignant diseases. It consist of three components:
a photosensitizer, laser light and oxygen. The procedure requires administration of a
photosensitizing agent followed by irradiation at appropriate wavelength. It result in generation
of reactive oxygen species (ROS), mainly singlet oxygen, which damage intracellular organelles
leading to the cell death. Antitumor effects of PDT result from direct cytotoxicity, damage to the
microvasculature, and induction of a local inflammatory reaction. Antivascular effect of PDT is
well-evaluated process, while lymphatic-specific PDT is a novel therapeutic strategy. It has been
demonstrated that verteporfin–PDT lead to specific lymphatic occlusion, however death
mechanism of lymphatic endothelial cells needs to be evaluated.
The aim of the study was to determine the death mechanism of lymphatic endothelial cells after
verteporfin–PDT.
All experiments were carried out using immortalized human lymphatic endothelial cells (LECs).
To perform PDT verteporfin and laser that emits the light of wavelength 690 nm were used.
Cytostatic/cytotoxic effects of the PDT was assessed by crystal violet cell viability assay.
Apoptosis, autophagy and endoplasmic reticulum stress processes were evaluated using Western
blotting, RT-PCR and flow cytometry methods.
Time- and dose-dependent PDT activity of caspase 3 was demonstrated by presence of its cleaved
form using Western blotting. Activity of this caspase was indirectly demonstrated by detecting 89
kD fragment of poly(ADP-ribose) polymerase (PARP). DNA fragmentation, one of the later step
in apoptosis, was measured 24h after PDT by TUNEL assay (ang. TransferasedUTP Nick End
Labeling) using flow cytometry. Spliced (s-XBP-1) and unspliced (u-XBP-1) forms of gene XBP1, a transcription factor involved in signal pathway in the endoplasmic reticulum stress, was
detected by RT-PCR and electrophoresis in agarose gel. To investigate the process of autophagy,
Western blotting was performed for evaluation of LC3-II form, protein associated with the
formation of autophagosomes.
159
THE INFLUENCE OF TUMOR DERIVED EXOSOMES IN HUMAN
REGULATORY T CELLS IN OVARIAN CANCER. PRELIMINARY
REPORT
Paulina Patalas1 , Marta Szajnik-Szczepański2
1
Department of Biochemistry, Nencki Institute of Experimental Biology PAN, Warsaw Department of Clinical
Immunology, Poznan University of Medical Sciences, Poznan
2
Department of Gynecology Oncology, Military Institute of Medicine, Warsaw, Poland Department of Gynecology
and Gynecologic Oncology, Poznan University of Medical Sciences, Poznan
Tumor-derived exosomes (TEX) are present in body fluid of patients with ovarian cancer (OvCa)
and might be involved in tumor progression. These small vesicles containing nucleic acid and
protein, perceived to be carriers of this cargo between many types of cells. The hypothesis is
tested that 1) OvCa cells as well as TEX contain ectonucleotidases CD39/ CD73 and ADA/CD26
2) TEX contribute to activation of human regulatory T cells 3) there is an association between the
exosomes presence/protein content in plasma of OvCa patients and disease outcome 3) TEX
contribute to conversion of CD4+CD25-CD39- into T regulatory lymphocytes.
Exosomes were purified from the supernatant of OvCa cell lines A2780 and SKOV3 and from the
plasma of patients with OvCa, benign tumor and healthy control using ultracentrifugation.
Exosomes were visualized by scanning electron microscopy and their protein content was
measured. The molecular profile was determined using Western blot. Ectonucleotidases
CD39/CD73 and ADA/CD26 expression in ovarian cancer tissues was evaluated by
immunohistochemistry. CD4+CD25+Foxp3 and CD4+CD25- cells were isolated from PBMC of
normal control and ovarian cancer patients and cultured with or without exosomes. Flow
cytometry was used to determine percentage and phenotype of the cells.
The OvCa patients‟ plasma contained higher level of exosomes (p<0,05) compared to those in the
plasma of benign tumor patients or normal control. Exosomes from plasma of subjects with OvCa
carried TGF-β, CD39, LAMP-1. High protein level of exosomes were seen in newly diagnosed as
well as less and more advanced patients. The exosomes levels variably changed in chemotherapy
treated OvCa patients. Immunohistochemical staining of human OvCa tissue specimens revealed
expression of CD39 in 70% and CD73 in 77% OvCa samples, whereas ADA and CD26 were
expressed in all samples. TEX convert CD4+CD25-CD39- into T regulatory lymphocytes.
This study suggests that TEX released by ovarian cancer cells activate T regulatory cells. This
represents a newly defined mechanism that might be involved in regulating peripheral tolerance
by tumors. Analyses of plasma exosomes levels offer a novel approach to diagnosis and
monitoring response to therapies in OvCa patients.
*This study was supported in part by the Foundation for Polish Science (ParentBridge
Program/2011/186) grants to MS.
160
SPHEROID-PLUG MODEL: A NEW TOOL IN CANCER RESEARCH
Monika Żukowska1, Krzysztof Szade1, Alicja Józkowicz1, Józef Dulak1
1
Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology,
JagiellonianUniversity, Krakow, Poland
The therapeutic potential of new anti-tumour drugs is evaluated by preclinical animal studies. However,
results obtained in animals often donot correlate with the effects obtained in clinical practice. This
discrepancy creates a demand for novel animal models of tumour development that will allow more
reliable drug screening. Here, we propose a spheroid-plug model that better reflects the complexity
ofcancer biology.
The spheroid-plug model is based on in vitro formation of spheroids from tumour cells. After 48 h, a
single spheroid (~250 µm diameter, 1500 cells) is injected into mouse subcutaneously within Matrigel.
This results in formation of Matrigel plug with a single tumour spheroid in contrast to classical model,
where cells are injected in suspension.
In our studies we used melanoma (B16F10) and lung cancer (LLC) cell lines that were stably transduced
with GFP and luciferase genes. Tumour development was monitored using 3-dimensional ultrasound (3D
USG) and non-invasive bioluminescence imaging (IVIS). Angiogenesis was measured with 3D USG with
PowerDoppler module. We performed all measurements at three time points: day 1, 7 and 14 after the
injection of tumour cells.
We observed a significant increase of tumour volume in B16F10 classical model in contrast to B16F10
spheroid-plug model at day 14 (14.01±9.02 vs. 1.03±0.38 fold, p=0.002). LLC tumours grown in classical
model were also bigger then LLC in spheroid-plug model, however the difference was smaller (2.69±1.11
vs. 1.82-fold±0.70 fold increase).
The tumour volumes assessed by 3D-USG did not correlate with IVIS measurements of bioluminescence.
We observed no differences in bioluminescent signal between classical and spheroid-plug model in
B16F10 and LLC tumours. As only viable cells reveal the enzymatic activity of luciferase, the observed
differences between the physical tumour size and bioluminescent signal may indicate the presence of not
viable necrotic areas.
To quantify the necrotic areas of tumours we introduced the Necrotic Factor (NF). NF is defined as the
ratio ofphysical tumour volume to IVIS signal. NF was much higher in B16F10 classical model than
spheroid model (2.92·10-8±1.42·10-8 vs. 6.56·10-9±1.78·10-7, p=0.0028) No statistically significant
differences were noted in LLC cell line (p=0.27). Additionally, necrotic changes in two models were
confirmed by histological analysis.
Next, we verified if augmented necrosis in tumours grown in classical model is caused by different
development of vessels. In classical model, there is an increase in percentage of vascularisation on day 7,
but it collapses until day 14: B16F10 (17.84±6.52% vs. 5.45±4.46%, p=0.0027), LLC (14.71±4.77% vs.
5.63±2.06%, p=.0.0011). Oppositely, in spheroid-plug model percentage of vasculature increases with
time in B16F10 (9.42±5.5% vs. 14.33±7.43%, p= 0.075) and only slightly decreases in LLC
(10.93±6.05% vs. 7.9±2.92%)
Concluding, tumour development and angiogenesis is different in classical and spheroid-plug model.
Spheroid-plug model is characterized by slower tumour growth, increased vascularisation and lower NF,
what better reflect the pathophysiology of cancer progression. These advantages make spheroid-plug
model a valuable tool for studies of tumour development and screening for new anti-tumour drugs.
161
RESTORATION OF ATG5 TO ATG5-DEFICIENT DU-145 CELLS BRINGS
BACK AUTOPHAGY, HOWEVER IT DOES NOT CHANGE SENSITIVITY
TO PHOTOPHRIN-MEDIATED PHOTODYNAMIC THERAPY
Domagała Antoni1, Gabrysiak Magdalena1, Barankiewicz Joanna1, Trzeciecka Anna,1 Firczuk
Małgorzata1
1
Department of Immunology, Medical University of Warsaw
Photodynamic therapy (PDT) is clinically approved and rapidly developing anticancer treatment.
Cytotoxic effects of PDT are mediated by reactive oxygen species generated via activation of
photosensitizer by visible light. Photodamage of cells can lead to induction of various cell death
types, like necrosis, apoptosis, autophagy. However, autophagy is a double-edged sword, having
both tumor-promoting and tumor-suppressing properties, therefore it is crucial to elucidate its
role in PDT in various experimental models.
The aim of this study was to investigate the role of autophagy in DU-145 cells subjected to in
vitro photophrin-mediated photodynamic therapy.
All experiments were performed on human-prostate cancer cell line – DU-145.
Cytostatic/cytotoxic effects were measured in crystal violet staining assay. Western blotting was
used to assess autophagy. GFP-LC3 and ATG5 protein expression was obtained by lentiviral
tranduction method.
Typical hallmarks of autophagy were neither detected in DU-145 cells upon PDT nor after
incubation with rapamycin - potent autophagy activator. Increase in LC3 puncta, attributed to
autophagy induction, was not observed in immunofluorescence analysis of DU-145 cells.
Western blot analysis revealed that there is no processing of LC3-I to its activated form - LC3-II.
However, both PDT and chloroquine led to substantial accumulation of LC3-I. These results
strongly suggest that DU-145 cell line is autophagy-deficient. It is in agreement with a recent
report by Ouyang et al., 2013, who reports that autophagic pathway is impaired in DU-145 cells
due to mutations in ATG5 gene. Protein encoded by this gene is critical for autophagosome
formation and LC3 conversion. Therefore, we introduced the wild-type ATG5 gene to DU-145
cells via lentiviral transduction. As assessed by Western blotting, ATG5 protein and ATG12ATG5 covalent conjugates were present in cells infected with viral particles encoding wild-type
ATG5. Photodynamic therapy led to induction of autophagy in DU-145 cells stably expressing
ATG5, which was confirmed by accumulation of LC3-II, a faithful marker of autophagy
stimulation. To investigate the role of autophagy, ATG5-expressing and ATG5-deficient DU145
cells were subjected to PDT. The experiment revealed that ATG5-deficient DU145 cells are
slightly more sensitive to photodynamic therapy.
These study demonstrates that ATG5 expression in DU-145 restores autophagy, however it
marginally affects the sensitivity of this cell line to photodynamic therapy.
162
LOOKING FOR A CURE TO DIABETES LYSOPHOSPHATIDYLCHOLINE
Anna Katarzyna Drzazga1
1
Institute of Technical Biochemistry, Lodz University of Technology
Diabetes mellitus is one of the most common metabolic diseases nowadays. The most often it
results from insulin deficiency due to destruction of islet cells in pancreas (type 1 diabetes, T1D)
or insulin resistance resulting from reduced number of insulin receptors and disruption of cellular
signaling pathways (type 2 diabetes, T2D). T2D counts for over 90 % of all cases of the disease
and affects over 340 million people worldwide. This also entails development of other disorders,
like heart diseases, strokes, obesity, diabetic retinopathy, kidney failure or even poor blood
circulation in limbs.
Due to high epidemiology rates of the disease and related severe effects, there exists a great need
to search for a successful method of treatment. A significant part of researchin this field is
focused on understanding of cellular signaling connected with regulation of insulin secretion.
Moreover, the ongoing development of nutrigenomics indicates the meaning of proper diet in
disease therapy and prophylaxis.
According to recent reports,the main metabolite of lecithin - lysophosphatidylcholine (LPC)is
one of the most efficient agonist of G-protein coupled receptors expressed in pancreas and gut,
responsible for modulation of insulin secretion, activation of glucose uptake and effective
decrease of blood sugar level. However, the molecular mechanisms behind these observations
have not been understood yet.
The following study is devoted to LPC and its analogues originally synthesized at the Institute of
Technical Biochemistry, Lodz University of Technology, Poland. The goal is to show their great
potential as novel therapeutics, based on preliminary in vitro investigation.
163
NEW USE OF OLD DRUG: VALPROIC ACID PROMOTES
RHABDOMYOSARCOMA CELL DIFFERENTIATION
M. Jeż1, M. Cieśla1, M. Kozakowska1, J. Dulak1, A. Józkowicz1
1
University, Krakow, Poland.
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma, mostly characterized by
early age of onset. We have shown recently that heme oxygenase (HO-1), a cytoprotective
enzyme degrading heme, is upregulated in RMS and that its inhibition promotes lower
aggressiveness of this tumour. Another protein strongly involved in RMS pathogenesis is Yin
Yang 1 (YY1) transcription factor. YY1 is known to inhibit class of muscle specific microRNAs
(myomiRs) of miRNA-29 family. In this manner YY1 abrogates terminal myogenic
differentiation.
According to our studies, both HO-1 and YY1 are expressed at higher levels in more aggressive
sub-type of RMS, termed alveolar RMS (aRMS) in comparison to more benign, embryonal RMS
(eRMS). We also identified a novel inhibitor of HO-1 expression – valproic acid (VA), known as
a histone deacetylase inhibitor, which is a drug commonly used for epilepsy treatment.
We have shown that VA downregulates expression of HO-1 both in vivo and in vitro, and
concomitantly can abrogate tumour progression in vivo when administered locally. Moreover, it
decreases expression of YY1 and affects some of the markers of myogenic differentiation.
Interestingly, despite diminishing YY1, VA reduces levels of myomiRs of miRNA-29 family and
it decreases also microRNAs targeting HO-1 3'UTR. On the other hand, VA upregulates the
hypoxia inducible factor1 (HIF1) and Bach1 repressor levels. As these factors diminish human
HO-1 expression, we propose that VA inhibits HO-1 by influencing HIF1/ Bach1 axis, thereby
overcoming effect of VA on miRNAs targeting HO-1. Via this process VA might promote more
differentiated state of RMS.
In conclusion, HO-1 may serve as a putative target for future therapies of RMS. VA, as a drug
already accessible in clinic, seems the promising molecule for such targeted therapies.
164
PHAGE THERAPY – MEDICAL APPLICATIONS AND PERSPECTIVES
Rajmund Królikowski1,2
1
Department of Microbiology; Faculty of Biochemistry, Biophysics and Biotechnology
2
Scientific Association of Biotechnology Students “Mygen”
As modern society approaches post-antibiotic era, multiple drug resistant bacterial strains pose
ever-increasing threat. Infectious diseases, which were the major cause of death until early XX
century, are having a comeback claiming more lives every year. But where antibiotics fail, a
nearly forgotten therapeutic steps in providing treatment in virtually every case of drug resistant
infection.
Bacteriophages have been successfully used to fend off pathogenic bacteria since 1919 and since
then numerous therapeutic phage strains have been isolated and used in treatment of various
infections, including methicillin-resistant Staphylococcus aureus (MRSA). Despite nearly a
century of scientific study and medical application phage therapy is still considered as
experimental, or as a treatment of last resort. However, recent studies prove, that phage therapy
can be more effective and less costly than a standard antibiotic treatment. Furthermore the
development of novel therapeutic phages is much easier and cheaper than that of conventional
drugs. As new commercially available phage preparations appear on the market, phage therapy
becomes more popular, and proves to have great potential to become a standard treatment in the
battle against pathogenic bacteria.
165
SYNTHESIS AND BIOLOGICAL ACTIVITY OF NEW GLUCOSAMINE-6PHOSPHATE SYNTHASE INHIBITORS.
Pawlak Dorota1,2; Stolarska Magdalena1,2; Andruszkiewicz Ryszard1;
1
Departament of Pharmaceutical Technology and Biochemistry,
2
Student‟s Society of Biotechnology Gdańsk University of Technology, Narutowicza 11/12, 80-233 Gdańsk, Poland
Studies in recent years show that the frequent use of antibiotics is potentially one of the main
reasons for the increased cases of fungal and bacterial infections, especially the most dangerous
systemic infections. So far, there are no chemotherapeutic that would give sufficiently
effective,withougt toxic effect to humans. Nowdays scientistc are looking for a potential
molecular target whitch would have applied for antifungal therapy. One of these targets that has
been proposed was glucosamine-6-phosphate (Gln-6-P) synthase.
Glucosamine-6-phosphate synthase is a key enzyme in the biosynthesis of aminosugar builder of
macromolecules included in the cell wall. This enzyme catalyzes the first step of hexosamine
metabolism. Gln-6-P synthase is located in pathogen and mamalian cell, but inactivation in
human cells, does not produce negative effects, and cause lysis of microbial cells.
Over the years there has been discovered many Gln-6-P synthase inhibitors. It can be expected
that the analogs of substrates will have the ability to inhibit this enzyme. One of the strongest
and specific inhibitors of Gln-6-P syntase is an analog of L-glutamine, i.e. N3-(4metoxyfumaroyl)-L-2,3-diamiopropanoic acids (FMDP). This compound however, exhibits weak
antimicrobial activity, due to poor transport into the microbial cells. Novel, selective and readily
transported of Glc-6-P synthase inhibitors may be usefull as a tool in managing fungal infections.
In my presentation I would like to present the designed and synthesized novel of FMDP
derivatives, problems with using them as chemotherapeutics and the relationship between the
structure and biological activity of the derivatives obtained.
In summary, the synthesis of novel inhibitors of Gln-6-P synthase increased the lipophilicity and
interaction with the active site of the enzyme, whitch provides an opportunity to improve the
biological activity of the compound. New, improved derivatives may be a chance to find new
effective antimicrobial chemotherapeutic agent, without the negative side effects for the human.
166
ADENOVIRUS VECTOR WITH MUSCLE-SPECIFIC EXPRESSION
CASETTEAND LAMIN A GENE FOR GENE THERAPY OF
LAMINOPATHIES
Anna Way1
1
Laboratory of Nuclear Proteins, University Of Wrocław
Laminopathies are rare genetic diseases caused by mutations either in the genes (LMNA,
LMNB1, LMNB2) encoding proteins of inner nuclear membrane, called lamins, or enzymes,
which are taking part in the post translational modifications of these proteins, for instance,
metalloprotease ZMPSTE24. Symptoms of these diseases differ, depending on mutation, but we
can distinguish three main types: muscular dystrophies, lipodystrophies and neuropathies. So far,
no cure or therapy has been developed to treat these diseases, but some are under investigation.
Gene therapy with the use of viral vectors is one of them. The main idea is to deliver the proper
gene to the cells so correct protein will be expressed. Gene of interest is carried in the viral
vector. One of the common viral vectors, which is right now under investigation, are
adenoviruses. Despite some disadvantages, adenoviruses are used due to their properties, like
high capacity of transgene, which allows inserting relatively long gene with regulatory sequence,
ability to infect a wide variety of cells, including both dividing and non-dividing cells and the
ability to be produced in high titers and easy manipulation. They are also not integrating with
host genome, which lowers the possibility of insertional mutagenesis.
During my Master program research, I will be producing an adenoviral vector with musclespecific expression cassette and lamin A gene for the gene therapy of laminopathies, as well as
vector expressing lamin A under the CMV promoter (depending on the progress and results other
variants are possible). Later on, biodistribution of produced proteins will be examined in vivo on
a mice model. Adenoviruses besides lamin A will express GFP. This will allow determining
which types of tissue are characterized with the highest and lowest expression of desired protein.
Production of viral vector will be conducted using an AdEasy system and HEK293FT cells.
167
SPICE PLANTS UNDER HEAVY METAL STRESS CONDITIONS
Kamila Kulbat1, Agnieszka Szczodrowska1
1
Politechnika Łódzka, Wydział Biotechnologii i Nauk o Żywności, Instytut Podstaw Chemii Żywności
During vegetation plants are exposed to many environmental pollutants such as presence of
heavy metal ions in the soil, chemical pesticides, excessive UV radiation or attack of pathogenic
microorganisms. One of the first defensive reactions of living cells is an increase in the
concentration of reactive oxygen species (ROS). To protect the living cells under stress
conditions plants begin to synthesize a series of defensive secondary metabolites, among which
the polyphenolic compounds with antioxidant activity and defensive proteins play the most
important role. Defensive proteins known as pathogenesis related proteins (PR proteins) exhibit
different antimicrobial activity and in the company of polyphenols form a defensive shield. Some
of PR proteins have documented allergenic properties and could cause severe allergic reactions,
including anaphylactic shock.
In our work we examined the concentrations of polyphenolic compounds in the tissues of popular
spice plants: basil (Ocimum basilicum), peppermint (Mentha piperita), oregano (Origanum
vulgare), parsley (Petroselinum crispum), dill (Anethum graveolens) and coriander(Coriandrum
sativum), their antioxidant properties as well as the concentration and identification of defensive
proteins. We compared the content of these secondary metabolites in control plants vs. plants
under heavy metal stress condition. During our research we were trying to check dependence
between plant breeding conditions and the actual contents of defensive metabolites. We were
trying to answer the question if there is any correlation between concentration of valuable
antioxidant compounds and the content of pathogenesis related proteins, which are a source of
potential allergens.
168
POLISH TRICHODERMA STRAINS - BIOLOGICAL CONTROL AGENTS
WITH PLANT PROTECTION POTENTIAL
J. Nawrocka1, U. Małolepsza2, M. Szczech3
1,2
3
Department of Plant Physiology and Biochemistry University of Łódź, Poland
Horticulture Research Institute Skierniewice, Poland
The subject of this study is associated with the current stream of investigations involving the
search for new, environmentally friendly methods of protecting plants against infectious diseases.
The use of beneficial microorganisms, such as fungi of the Trichoderma strains, can help to
reduce the use of harmful chemical pesticides. The possitive influence of Trichoderma is
associated with stimulation of plant growth and biomass production. In recent years more
attention has been paid to the potential ability of Trichoderma strainsto activate natural defense
response reinforcing the resistance of plants to diseases caused by pathogens. However, the type
and biochemical basis of Trichoderma- induced resistance in plants are poorly understood and
require clarification.
Consequently, the main objective of this study was associated with the current trend of
research leading to determination of the type of biochemical reactions and resistance induced by
Trichoderma. The impact of twenty five Polish Trichoderma strains added to the growing
medium on seed germination and growth of cucumber plants (Cucumis sativus) cv. Iwa F1 was
tested. Furthermore, the objective was to determine the effect of selected strains on induction of
plant defense reactions to infection by Rhizoctonia solani, pathogenic fungi causing significant
losses in cucumber plant crops. The changes in phenylalanine ammonia lyase (PAL) activities as
well as in total phenolic (TP) and ortodihydroxyphenolic(oDP) compound concentrations in
cucumber leaf tissues were studied as symptoms associted with induction of plant defense
reactions and resistance.
In this study two strains of Trichoderma: T. atroviride TRS 25 and T. virens TRS 106
significantly increased cucumber seed germination, plant growth and biomass production. When
their spores were present in the growth medium plant root infection by the pathogen R. solani
was supressed. Parallelly, increases in PAL activity and in TP and oDP compound concentrations
both in healthy and R. solani-inoculated plants were observed which may suggest that
Trichoderma strains supressed the pathogen infection due to their ability to mobilize natural
defense response and resistance in plants related i.a. to increased synthesis of phenolic
compounds.
The present results encourage further analysis of the selected Trichoderma strains as they seem to
have a potential to promote plant growth and to induce resistance to
R. solan, which can be used in the future for plant protection in Polish agriculture.
This work was supported by grant no: UDA-POIG.01.03.01-00-129/09-04 (POIG.01.03.01-00-129/09-05: “Polish
strains of Trichoderma sp. in biocontrol and to make productive organic waste”).
169
THE USE OF PALMARIA PALMATA ALGAE IN THE ACTIVE
BIOMONITORING OF THE SURFACE WATERS
Maria Zielińska1, Małgorzata Rajfur1, Andrzej Kłos1
1
Independent Chair of Biotechnology and Molecular Biology, OpoleUniversity
Biomonitoringis defined as the use ofbiological materialforevaluation andobservation ofthe
changes in the environmenttaking placeunder the influence ofvarious factors,
bothnaturalandanthropogenic. Information obtainedin this wayareused for exampleinthe flowing
waterqualitycontrol program.
Organisms living in the environment are constantly exposed to the physical, biological and
chemical influences. Organisms that have a tendency to accumulate chemicals can often
accumulate significant quantities of material from very low concentrations in the environment,
that`s why they are an useful tool in biomonitoring. Mosses, algae and lichens have been used by
many investigators to monitor heavy metal concentrations in waters and air because of their
tendency to selectively adsorb heavy metals.
Thealgae used in the study,Palmariapalmata,are widespreadin thecold waters ofthe North
Atlanticandthe Arctic Ocean, but also in the warmerwatersaroundPortugal,Spain andNew Jersey.
They occur at a depthof about 20meters andareknown for theirnutritional valueandfor high
content ofantioxidants. They are a good source of minerals and vitamins compared with other
vegetables, contain all trace elements needed by humans and have a high protein content.
The objective of thisstudywas an activebiomonitoring of the 8.5 kmsection of the Odra
riverwithin theOpole city limits. The studyusedmarine algae Palmariapalmata. Afterthe exposure
period, thealgae were analysed using atomic absorption spectrometry(AAS) to define the
concentrations ofheavy metals – Mn, Fe, Cu, Pb, Znand Hg. Metals were also determinedin water
samples.
Among thedetermined metalsthegreatestconcentrationswere characterized byiron and manganese
in the whole analysed river section. Designatedrelativeaccumulationfactors(RAF) indicate a
heterogeneous heavy metal contamination ofthe water.The largestincreases inconcentrations of
heavy metalsin exposedalgae sampleswere determined inthe harbor of theMetalchemEstate but
there was no concentration increase of copper and lead. The resultsindicate agoodaccumulationof
algaePalmariapalmata.
170
POLISH TRICHODERMA STRAINS - BIOLOGICAL CONTROL AGENTS
WITH PLANT PROTECTION POTENTIAL
J. Nawrocka1, U. Małolepsza2, M. Szczech3
1,2
3
Department of Plant Physiology and Biochemistry University of Łódź, Poland
Horticulture Research Institute Skierniewice, Poland
The subject of this study is associated with the current stream of investigations involving the
search for new, environmentally friendly methods of protecting plants against infectious diseases.
The use of beneficial microorganisms, such as fungi of the Trichoderma strains, can help to
reduce the use of harmful chemical pesticides. The possitive influence of Trichoderma is
associated with stimulation of plant growth and biomass production. In recent years more
attention has been paid to the potential ability of Trichoderma strainsto activate natural defense
response reinforcing the resistance of plants to diseases caused by pathogens. However, the type
and biochemical basis of Trichoderma- induced resistance in plants are poorly understood and
require clarification.
Consequently, the main objective of this study was associated with the current trend of
research leading to determination of the type of biochemical reactions and resistance induced by
Trichoderma. The impact of twenty five Polish Trichoderma strains added to the growing
medium on seed germination and growth of cucumber plants (Cucumis sativus) cv. Iwa F1 was
tested. Furthermore, the objective was to determine the effect of selected strains on induction of
plant defense reactions to infection by Rhizoctonia solani, pathogenic fungi causing significant
losses in cucumber plant crops. The changes in phenylalanine ammonia lyase (PAL) activities as
well as in total phenolic (TP) and ortodihydroxyphenolic(oDP) compound concentrations in
cucumber leaf tissues were studied as symptoms associted with induction of plant defense
reactions and resistance.
In this study two strains of Trichoderma: T. atroviride TRS 25 and T. virens TRS 106
significantly increased cucumber seed germination, plant growth and biomass production. When
their spores were present in the growth medium plant root infection by the pathogen R. solani
was supressed. Parallelly, increases in PAL activity and in TP and oDP compound concentrations
both in healthy and R. solani-inoculated plants were observed which may suggest that
Trichoderma strains supressed the pathogen infection due to their ability to mobilize natural
defense response and resistance in plants related i.a. to increased synthesis of phenolic
compounds.
The present results encourage further analysis of the selected Trichoderma strains as they seem to
have a potential to promote plant growth and to induce resistance to
R. solan, which can be used in the future for plant protection in Polish agriculture.
This work was supported by grant no: UDA-POIG.01.03.01-00-129/09-04 (POIG.01.03.01-00-129/09-05: “Polish
strains of Trichoderma sp. in biocontrol and to make productive organic waste”).
171
COMMERCIALLY AVAILABLE APPLICATIONS OF SELECTED
BIOTECHNIQUES VARIETY IN THE CAPTIVE BRED REPTILE
INDUSTRY BASED ON EXAMPLE OF PYTHON REGIUS (SHAW 1802)
COLOR MORPHS
Michał Skawiński
Uniwersytet Wrocławski
As the ownership and husbandry of exotic pets becomes more and more popular hobby, captive
bred reptile industry expands each year. According to The Modern U.S. Reptile Industry report by
Ariel H. Collis, M.A. Robert N. Fenili, PhD, only within the United States of America yearly
revenues of businesses that sell, provide services and manufacture products for reptiles, can
exceed $1.0 billion total. Some of the most popular and established species in hobby (i.e. Python
regius, Shaw 1802) were already put through traditional means of selective breeding, resulting in
specimens that feature traits or group of traits, desired and potentially highly priced by hobbyists.
Although various uses of biotechniques in producing captive bred Python regius (Shaw 1802)
color morphs may seem obvious, they are generally absent in the process. This work tries to give
a glimpse of possibilities that modern biotechnology can offer to the captive bred reptile industry,
based on the example of Python regius (Shaw 1802) color morphs.
172
DYNAMIC INSTABILITY EVENTS IN MICROTUBULE OVERLAPS IN
VITRO
Piotr Topolewski1, Aniek W. Jongerius2 Marcel E. Janson2
1
Warsaw University of Life Science, Poland
2
Wageningen University and Research Centre, Laboratory of Cell Biology, The Netherlands
Antiparalell microtubules (MTs) within the mitotic or meiotic spindle form overlaps, which are
involved in spindle pole separation and maintenance of the spindle bipolarity. In general, the
overlap serves as a place, where many motor proteins and regulators of microtubule dynamics
perform their function. To create such an overlap, a crosslinking protein, e.g. Ase1 is required.
To fully understand mechanisms that rule spindle assembly and maintenance, an elucidation of
the role of particular motor and non motor proteins occurring in overlaps has to be performed.
However,in vivo models have some disadvantages e.g. not sufficient separation of investigated
factors and limited possibilities of gene knock out methods. For these reasons, application of in
vitro assays seems to be necessary.
Previously developed in vitro models ensure investigationof either sliding properties of different
motors, or influence of non-motor proteins on the dynamic behavior of a single microtubule. In
this work, we show a new in vitro assay, enabling to study the influence of microtubule
associated proteins (MAPs) on the polymerization and depolymerization events (named dynamic
instability events) of overlapping MTs. Our assay relies on imaging of proteins bound to a
growing overlap. It is obtained by creating the overlap on a glass surface using Ase1, stimulation
of the microtubule dynamic instability and finally observation of the protein behavior.
The first protein investigated in our assay was Ase1 itself. We observed, that Ase1 very precisely
follows an overlap during dynamic instability events, what give us hope for further successful
studying functions of other proteins, such as CLASP or KLP9.
173
BIOACTIVE COMPOUNDS IN COFFEE BEANS AND THEIR
BENEFICIAL EFFECT ON HUMAN HEALTH
Donata Izabela Zaczyńska, Małgorzata Irena Bojczuk
Politechnika Łódzka, Wydział Biotechnologii i Nauk o Żywności, Instytut Chemicznej Technologii Żywności,
Zakład Technologii Skrobi i Cukiernictwa
Coffee, one of the most popular beverages, is consumed by millions of people every day. As the
most desired values include its taste and aroma, not everyone may know that coffee has also
beneficial influence on human health. This is mainly due to presence of antioxidants naturally
occurring in coffee beans. Those compounds inhibit oxidation of other molecules. Antioxidants
are very important elements of human diet, responsible for maintenance of redox homeostasis.
Due to high antioxidants content coffee intake is associated with reduced risk of different
pathological changes in the humanbody, resulting in premature aging and numerous diseases,
such as different types of cancers, brain dysfunctions (Parkinson‟s and Alzheimer‟s diseases) and
diabetes, etc.Compared to other drinks, coffee isconsidered to be one of the greatest sourcesof
antioxidants, polyphenols, and other biologically active compounds. One of the discussed issues
will concern antioxidants absorption into the human bloodstream.
Antioxidant activity of coffee (ability to inhibit the process of oxidation) results mainly from
presence of compounds such as: chlorogenic, ferulic, caffeic, and n-coumaric acids. In some
publications caffeine is also considered to be an antioxidant. It should be not forgotten that
antioxidant activity depends on different factors, such as variety of coffee, geographical area of
cultivation, roasting process parameters, as well as, ways of coffee brewing. The goal of this
presentation is to compare recent studies on antioxidant activity, depending on above mentioned
parameters. According to literature, Guatemala, Nicaragua and Colombia may be indicated
among other countries producing coffee with the highest antioxidant potential. Comparison of
scavenging of ABTS radical anions by coffees originating from mentioned countries with the
activity of some standards, including typical coffee compounds and common food additives will
be presented. Additionally, comparison between the antioxidant activity of coffees brewed in
different ways (espresso, instant and extract) will be discussed.
References:
1. Yashin, A., Yashin, Y., Wang, J.Y., Nemzer, B. (2013). Antioxidant and Antiradical
Activity of Coffee, Antioxidants 2, 230-245.
2. Parras, P., Martínez-Tomé, M., Jiménez A.M., Murcia M.A. (2007). Antioxidant capacity
of coffees of several origins brewed following three different procedures, Food Chemistry
102, 582-592.
3. Esquivel, P., Jiménez, V. M. (2012). Functional properties of coffee and coffee byproducts, Food Research International 46, 488-495.
174
INTEGRATIVE METHODOLOGY FOR THE IDENTIFICATION OF
ANTIVIRAL DRUGS AGAINST INFLUENZA PROTEIN TARGETS
Teresa Domaszewska1,2, Daniel A. Engel1, Zygmunt S. Derewenda1
1
University of Virginia
2
Wrocław University of Technology
Influenza is associated with significant morbidity and mortality and poses a continuing
worldwide public health problem. Anually, the virus causes 3 to 5 million cases of severe illness
and up to 500,000 human deaths in seasonal epidemics. Occasional outbreaks of influenza
pandemics represent a global threat.
Existing antiviral therapies against influenza are based on adamantanes and neuraminidase
inhibitors. However, widespread resistance is a growing concern for their continued clinical use.
The seasonal vaccine presents variable efficacy from year to year, due to constant antigenic drift
between the virus strains. Hence, there is an urgent need for the development of antivirals that
can be used both therapeutically and prophylactically.
The highly conserved NS1 and NP proteins are critical for the efficient expression of viral genes
and response to host defense mechanisms. This study presents a novel approach for the
development of drugs which target NS1 and NP from the influenza H1N1 and H3N2 strains. The
method enables the identification of compounds which physically interact with viral proteins and
have a robust anitiviral effect.
Our methodology integrates antiviral, microbiological, biochemical, and biophysical assays.
First, we screen small molecule libraries for antiviral activity in a yeast-based system. Yeast
expressing viral proteins have a slow-growth phenotype, which is reversed by the addition of a
potent antiviral. To limit off-target effects we validate the antiviral effect in mammalian cells and
test for direct physical interaction with NS1 or NP using thermal shift assay and dynamic light
scattering. The positive hits are further characterized by isothermal titration calorimetry and are
set up for X-ray crystallography studies. The proposed methodology is generic and can be used
against proteins from other viruses. We demonstrate this by studying interactions between the
Dengue NS3 protease and it‟s inhibitor, aprotinin.
175
TALE-TFs as gene therapy tool. Applications in Congenita dysceratosis
treatment
Michał Rudnik
Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Kraków, Poland
mi.rudnik@uj.edu.pl
Gene therapy is one of the most dynamic developing area in biomedicine and biotechnology.
Recently published data showed that originating from Xanthomonas TALE domains could be a
promising tool in cell and gene therapy. This proteins, which could be easily designed, consist of
dozen repeated units which allow to specifically recognize DNA sequence. Until today many
modifications of TALE domains are available, in which DNA binding domain is combined with
nucleases, recombinases or transactivation domains.
Together DNA binding domain with transactivation unit are called TALE-TFs. Thank to that
combination these proteins could be tailored to different promoters‟ sequences and specifically
activate or significantly enhance gene expression. This approach was used in researches focusing
on Congenita dyskeratosis therapy.
Congenita dyskeratosis is rare progressive disorder, mainly affecting skin and organ‟s protecting
layers. Similarly to progeria, it resembles premature aging, but the most serious complication is
bone marrow failure. Pathology of the disorder is not fully understood, but poor telomeres
maintenance was observed. What is more, overexpression of RNA component of telomerase
(TERC) resulted in keratinocytes correction and general health improvment.
In this studies we wanted to design several TALE-TF with potential to accelerate TERC
expression in firstly in cell lines and in future in iPS model.
176
EXPRESSION OF CCR3 AS A SINGLE BASOPHIL IDENTIFICATION
MARKER
Anna Skotny1, Barbara Kmiecik2, Ewa Zbrojewicz1, Katarzyna Dudziak3
1
Wroclaw Medical University, Department of Internal Diseases, Geriatrics and Allergology, Wybrzeze L. Pasteura
4, 50-367 Wroclaw, Poland
2
Wroclaw University of Technology, Institute of Materials Science and Applied Mechanics,
ul. Smoluchowskiego 25, 50-370 Wroclaw, Poland
3
Wroclaw Medical University, Laboratory of Neurotoxicology and Environmental Diagnostics, , ul. Bartla 5, 51-618
Wroclaw, Poland
Allergy is one of the chronic diseases that greatly reduces the quality of patients‟ lives. We
observe the growing number of people suffering from different types of hypersensitivities. There
are several, rare but potentially life-threatening complications of allergic diseases. Nowadays,
diagnostics offers a wide range of improvements. One of the very promising techniques is flow
cytometry (FCM). The combination of biotechnology, optics, electronics and advanced statistics
present in FCM, provides a qualitative and quantitative characterization of the examined
materials. There is a great need of finding a method in which no risk of causing anaphylaxis or
other dangerous complications, as in todays in vivo food, drug or insect venom allergy diagnosis
may occur.
Basophil activation tests (BAT) meet all of these expectations. Basophils were discovered 150
years ago by Paul Erhlich. Since then, they were not considered as being important (as
lymphocytes or other blood cells). Recently, we observe a growing interest in BAT. The
aforementioned method is time-efficient and enables an accurate and selective diagnosis of a
patient. Still, there is much to be done in the area of the new markers used in the identification
protocols. Thus, existing protocols are not ideal. The conducted research proves the potential of
anti-CCR3 and recognizes it as a great identification marker. Nevertheless, further research needs
to be done. It would be highly recommended to conduct a comparative research concerning the
protocols as follows: CCR3+, CRTH2+/CD3-/CD203c+ and CD123+/HLA-DR- [1]. Such
research would distinguish the most stable and accurate protocol of identification, as well as
would serve as a trigger for further research for finding the new markers that remain
undiscovered.
However, due to the lack of well-established standard procedures, the method is yet considered as
an inappropriate for routine diagnostics. Properly selected activation markers and identification
protocols make BAT test to be considered as an excellent complementary diagnostic method. In
conclusion, making BAT a standard in allergy diagnostics could largely improve patients‟ comfort
and safety.
References
[1] A. Wolańczyk-Mędrala, W. Barg, W. Mędrala, CD164 as a Basophil Activation Marker
Current Pharmaceutical Design, 17, 3786, (2011).
177
WHAT DO WE KNOW ABOUT RHEUMATOID ARTHRITIS?
Joanna Kałafut, Małgorzata Płonka
Uniwersytet Marii Curie-Skłodowskiej w Lublinie
Rheumatoid arthritis (RA) is an autoimmune chronic conditionconcerning thewhole organism. It
affects up to 1% of the world population, regardless ethnicity or age, but with four times higher
prevalence in females.
During the disease there are periods with acute symptoms and remission. There is a broad
spectrum of clinical manifestations of RA which includes swelling, pain and stiffness of the
altered joint. The pathogenesis mechanism remains largely unknown, although many researches
pointed out some determinants of disease development:
-socioeconomic factors (social class, educational level, lifestyle –e.g. smoking, diet),
-other diseases or family history of RA,
-microbial or viral infections,
-genetic markers (human leucocyte antigen (HLA)-DRB1 shared epitope(SE), the protein
tyrosine
phosphatase
(PTPN21)1858T
variant,
anti-citrullinated
protein
antibodies(ACPAs)and immunoglobulinM-rheumatoid factor(IgM-RF)).
The main aim of the treatment is to capture the inflammation process and to prevent tissue
damage. The therapy is combinespharmacotherapy, diet, physiotherapy and surgery.
The publication describesgeneral mechanisms of the RA pathogenesis, with emphasis on the steps
that could be the target of personalized therapy. It shows correlation between the genetic,
pathophysiological and environmental factors that result in variation of response.
178
Author index
A
Adamek E. ......................................................................... 57
Adamus T. .......................................................................... 17
Adamus-Białek W. ............................................................. 66
Agier J. ......................................................................... 13, 35
Albrecht J. ........................................................................ 105
Andruszkiewicz R. ............................................................ 166
Andrzejczak O. ................................................................... 14
Anisiewicz A. .............................................................. 15, 136
Antoszewski M. ................................................................. 16
B
Badyra B. ........................................................................... 17
Balcerzak M. .................................................................... 132
Banach M. ................................................................... 18, 50
Baocyr E. ............................................................................ 19
Barankiewicz J. ................................................................ 162
Baranowska A. ................................................................. 151
Baraoska K. ........................................................................ 20
Barszcz B.......................................................................... 102
Bartnik E. ........................................................................... 46
Bartosiak M. ................................................................ 21, 22
Bartosik D. ..................................................... 79, 82, 99, 156
Bazan J. ................................................................ 37, 51, 154
Bąk M. ............................................................................... 23
Bełczącka I. ........................................................................ 24
Bereta J. ....................................................................... 62, 67
Bernat P. ...................................................... 38, 93, 112, 149
Biała W. ............................................................................. 25
Białasik D. ........................................................................ 117
Białkowska A. .................................................................... 59
Bierwagen P....................................................................... 25
Bilska S............................................................................... 55
Boguszewska K. ................................................................. 26
Bojczuk M. I. .................................................................... 174
Borek I. .............................................................................. 77
Borowczyk J. ...................................................................... 76
Boryo M. ............................................................................ 27
Bzowska M. ....................................................................... 67
C
Chmielewska M. ................................................................ 28
Chojnacki M. .............................................................. 29, 147
Chołbioski P. .................................................................... 132
Chrzanowski M. ............................................................... 119
Chudzik B. .......................................................................... 34
Cierniak A. ......................................................................... 76
Cieśla M. .......................................................................... 164
Cieśla P. ..................................................................... 15, 136
Cieślak A. ..................................................................... 21, 22
Crosera M. ......................................................................... 76
Cybulski K. ................................................................. 30, 131
Czaczyk K. .................................................................... 36, 91
Czapla K. ............................................................................ 31
Czarnecki J. ............................................................ 79, 82, 99
Czarnek M. ........................................................................ 62
Czauderna S. ...................................................................... 62
Czemierska M. ................................................................... 32
Czerniak A. ................................................................... 84, 95
Czerwonka A. ....................................................... 33, 42, 152
Czerwonka G. ................................................ 47, 54, 65, 102
Czubatka A. ...................................................................... 107
Czuryło A. .......................................................................... 34
Czyż J. ........................................................................ 77, 141
D
Dadura K. ..................................................................... 13, 35
Dąbrowska N. .................................................................... 36
Demkow U. ........................................................................ 46
Derewenda Z. S................................................................ 175
Długooski J. ...................38, 45, 57, 93, 97, 98, 106, 112, 118
Dołhaoczuk-Śródka A. ....................................................... 48
Domagała A. .................................................................... 162
Domaszewska T. .............................................................. 175
Doolittle F. R. ..................................................................... 41
Drobna M. ......................................................................... 36
Drożdżyoska A. .................................................................. 36
Drukala J. ........................................................................... 76
Drzazga A. K. .................................................................... 163
Dudziak K. .................................................... 37, 51, 154, 177
Dudzik M. .................................................................. 38, 112
Dulak J. ................................................ 23, 72, 134, 161, 164
Dunajczyk N. ...................................................................... 39
Durak A. ............................................................................. 40
Dzierżawska J. .................................................................... 39
Dziewit Ł. ................................................................... 99, 156
Dziubak K. .................................................................... 21, 22
Dzwonkowska E. ................................................................ 36
E
Engel D. A. ....................................................................... 175
179
F
I
Fedorowicz J. ................................................................... 153
Felcenloben I. .................................................................... 19
Felczak A. ......................................................................... 149
Filipiak M. ........................................................................ 108
Filon F. L. ........................................................................... 76
Fiołka M. ............................................................................ 55
Firczuk M. ........................................................................ 162
Florczak J. .................................................................. 15, 136
Florczak T........................................................................... 59
Florczyk U. ....................................................................... 134
Foiani M........................................................................... 100
Fornal M. ........................................................................... 41
Frant M. ......................................................... 33, 42, 43, 152
Frasioski S. ......................................................................... 44
Frączak S. ........................................................................... 45
Frączyk J. ........................................................................... 49
Frycz B. .............................................................................. 74
Iwanaszko M.................................................................... 104
G
Gabrysiak M. ................................................................... 162
Gagoś M. ........................................................................... 34
Gawlik-Dziki U. .......................................................... 40, 111
Ghanim M. ......................................................................... 46
Glazioska P. ....................................................................... 18
Gmiter D. ........................................................................... 47
Gniłka R. ............................................................................ 19
Godyo P. ............................................................................ 48
Gonciarz W. ....................................................................... 49
Goralczyk A. ....................................................................... 76
Gorzelaoczyk A. ................................................................. 50
Gostomska K........................................................ 37, 51, 154
Górecki A. ........................................................................ 151
Grabowska J....................................................................... 53
Gracz J. ............................................................................ 130
Grochot-Przęczek A. .......................................................... 72
Grudzieo M. ............................................................... 52, 158
Grządziel J. ....................................................................... 101
Grzemski A......................................................................... 53
Guzy A. .............................................................................. 54
H
Haliniak J. .................................................................. 15, 136
Hodorowicz M. ................................................................ 102
Hordyjewska E. .................................................................. 55
Hyra K. ............................................................................... 56
J
Jabłooska-Wawrzycka A. ................................................. 102
Jagielski J. .......................................................................... 68
Jagodzioski P. ..................................................................... 74
Jakubczak M. ................................................................... 104
Jamruszka T. ...................................................................... 89
Janczarek M. .................................................................... 101
Janicki T. ............................................................................ 57
Janik M. ............................................................................. 58
Janson M. E...................................................................... 173
Jarosz-Wilkołazka A. .......................................................... 32
Jarych D. ............................................................................ 59
Jaźwa A. ............................................................................. 23
Jendraszak M. .................................................................... 28
Jeż M. .............................................................................. 164
Jędroszkowiak A. ............................................................... 16
Jędrzejczyk I. .............................................................. 20, 128
Jongerius A. W. ................................................................ 173
Jotko M. ................................................................. 60, 61, 81
Józkowicz A.......................................... 23, 72, 134, 161, 164
Jóźwiak K. .......................................................................... 46
Jucha J. .............................................................................. 62
Jura J. ................................................................................. 76
Juszkiewicz S. ..................................................................... 62
K
Kabacioska M. ................................................................... 63
Kaca W............................................................. 47, 49, 54, 65
Kaczor J. ............................................................................. 33
Kadzewicz K. ...................................................................... 64
Kałafut J. .......................................................................... 178
Kałuża K. ............................................................................ 65
Kamioska E. ....................................................................... 66
Kamioski D. ........................................................................ 34
Kamioski Z. ........................................................................ 49
Kantyka T. ........................................................................ 113
Karabasz A. ........................................................................ 67
Karoo K. ....................................................................... 63, 68
Karp K. ............................................................................... 69
Kasprzyk A. ........................................................................ 43
Kawka K. ...................................................................... 21, 22
Kępska D. H........................................................................ 70
Kęsy J. .......................................................................... 18, 71
Klein A. .............................................................................. 76
Kliszcz B. ............................................................................ 55
180
Klóska D. ............................................................................ 72
Kłos A. ........................................................................ 48, 170
Kłossowski S. ................................................................... 120
Kmiecik B. ................................................................ 155, 177
Knobloch P......................................................................... 73
Kodroo A............................................................................ 46
Kolesioska B. ...................................................................... 49
Kołodziej P. ........................................................................ 55
Kołodziejczak A. ................................................................. 74
Kołodziejczyk I. .................................................................. 75
Komorowska M. .............................................................. 155
Kondrat A. ......................................................................... 16
Konieczna I. ............................................................... 49, 142
Konieczny P. ................................................................ 17, 76
Kopcewicz J........................................................................ 18
Kordowska-Wiater M. ..................................................... 144
Kosioska M. ............................................................... 77, 141
Kostrzewa-Susłow E........................................................... 19
Koszałkowska M. ............................................................... 78
Kotwicka M........................................................................ 28
Kowalska M. ...................................................................... 91
Kowalski Ł. ......................................................................... 79
Kozakowska M. ................................................................ 164
Koziel J. .............................................................................. 76
Kręcidło Ł. .......................................................................... 80
Królikowski R. .................................................................. 165
Krupioski M. ...................................................................... 57
Krzyśko-Łupicka T. ................................................. 31, 78, 80
Kubiak K. ............................................................................ 85
Kudko A.............................................................................. 18
Kudawska B. .......................................................... 60, 61, 81
Kulbat K. .......................................................................... 168
Kuźmicz K........................................................................... 82
Kwiatkowska D. ............................................................... 132
Kwinkowski M. .................................................................. 49
L
Laczna E. ............................................................................ 76
Langwioski W..................................................................... 83
Lasek R............................................................................. 156
Lesiecki M. ................................................................... 84, 95
Lewandowski M................................................................. 85
Lisowska K. .............................................................. 146, 149
Lorentowicz M. .................................................................. 86
M
Maciąg K. ........................................................................... 87
Maciejewska E. ................................................................ 157
Majewska I. ..................................................................... 127
Majka M. ........................................................................... 17
Makuch K. .......................................................................... 88
Małolepsza U. .......................................................... 169, 171
Marciniak K. ................................................................. 18, 50
Marzec A.......................................................................... 101
Matusiak R. ...................................................................... 127
Matysik G................................................................... 42, 152
Mazurkiewicz M. ............................................................. 128
Mazurkiewicz N. .......................................................... 89, 91
Michalik M. ................................................................ 77, 141
Michnicki P. ..................................................................... 106
Moryl M. ............................................................................ 35
Muchowicz A. .......................................................... 120, 159
N
Narożna B. ......................................................................... 90
Nawrocka J. ............................................................. 169, 171
Nicpoo S. ........................................................................... 92
Nowacki K. ......................................................................... 83
Nowak W. .......................................................................... 62
Nowogórska A. ................................................ 115, 116, 139
Nykiel J. ............................................................................. 93
O
Odrobioska J. ..................................................................... 58
Okła K. ....................................................................... 15, 136
Olejnik Ł. ............................................................................ 94
Olejnik P. ........................................................................... 63
Olejnik P. P. ................................................................. 84, 95
Olszewski B. ....................................................................... 96
Olszówka M. ...................................................................... 87
Olsztyoska-Janus S. .......................................................... 155
Orzełowska M. ........................................................... 15, 136
Osiak A. ............................................................................ 159
P
Pachla A. .................................................................... 29, 147
Paduch R. ..................................................................... 42, 43
Pałasz A. .......................................................................... 103
Pasioski M........................................................................ 128
Patalas P. ......................................................................... 160
Patykowski J. ................................................... 115, 116, 139
Pawlak D. ......................................................................... 166
Pawlicka J. ......................................................................... 91
Piątek M. A. ......................................................... 97, 98, 118
Piechowiak W. ................................................................... 68
Pięt M. ....................................................................... 29, 147
Piña M. ............................................................................ 100
181
Płaza K. ............................................................................ 113
Płonka M. ........................................................................ 178
Pokrywka A. ..................................................................... 132
Popławski T...................................................................... 107
Posmyk M. M. .................................................................... 75
Potempa J. ....................................................................... 113
Prochwicz E. ...................................................................... 99
Przetocka S. ..................................................................... 100
Pyza E. ............................................................................... 76
R
Rachwał K. ....................................................................... 101
Rajfur M........................................................................... 170
Rakicka M. ................................................................. 30, 131
Rakowiec M. ...................................................................... 75
Reiter J. .............................................................................. 25
Relich I. .............................................................................. 49
Rodzik O. ......................................................................... 148
Rogala P. .......................................................................... 102
Rojczyk-Gołębiewska E. ................................................... 103
Rolnik B............................................................................ 104
Różalska S. ......................................................... 45, 106, 118
Różycka A. ......................................................... 45, 105, 106
Rudnik M. ........................................................................ 176
Rusinek P. ........................................................................ 140
Ryszawy D.......................................................................... 77
Rzeski W. ........................................................................... 33
Smolarz A......................................................................... 117
Soboo A. .......................................... 38, 45, 93, 98, 112, 118
Stachecka J. ..................................................................... 119
Stachura J. ....................................................................... 120
Stapurewicz D. ................................................................. 121
Staśko M. ......................................................................... 122
Stelmaszczyk-Emmel A. ..................................................... 46
Stobioska M. .................................................... 123, 124, 133
Stolarska M. ..................................................................... 166
Sułkowski M. ..................................................................... 17
Szade K. ........................................................................... 161
Szajnik-Szczepaoski M. .................................................... 160
Szczech M. ............................................................... 169, 171
Szczepanowicz K. ............................................................... 67
Szczepaoska D. ................................................................ 125
Szczepaoska I. .................................................................. 126
Szcześ A. ............................................................................ 32
Szczodrowska A. .............................................................. 168
Szczuka E. .......................................................................... 43
Szeliga M. ........................................................................ 105
Szewczyk R. ........................................................................ 75
Szmyd R. ............................................................................ 76
Szpotan J.......................................................................... 127
Szpott D. .......................................................................... 128
Szustak M. ....................................................................... 129
Szymkowiak J. .................................................................. 130
Ś
S
Świeca M. ........................................................................ 111
Sakowicz T. ......................................................................... 44
Salachna D. ...................................................................... 143
Sarnik J. ........................................................................... 107
Sawa K. ............................................................................ 108
Seczyoska M. ................................................................... 109
Seliga A. ........................................................................... 110
Serafin W. ........................................................................ 108
Sęczyk Ł. .......................................................................... 111
Shubassi G. ...................................................................... 100
Siewiera P. ................................................................. 38, 112
Sitarska A. ........................................................................ 113
Sitarz K. ............................................................................ 114
Skalniak Ł. .......................................................................... 76
Skawioski M. .................................................................... 172
Skibioska I. ......................................................................... 28
Skotny A. ........................................................... 37, 155, 177
Skwarek M. ...................................................... 115, 116, 139
Słaba M.............................................................................. 93
Sładek K. ............................................................................ 23
Smeds E. ............................................................................ 41
T
Tomaszewska L. ......................................................... 30, 131
Tomela K. ........................................................................... 74
Tooska K. ........................................................................... 46
Topolewski P. .................................................................. 173
Tretyn A. ............................................................................ 18
Trzebuniak K. F. ............................................................... 132
Trzeciecka A. .................................................................... 162
Twardowski T. ................................................................. 130
Tyczewska A. ................................................................... 130
U
Urbaniak P. ........................................................................ 28
Urbaoski D. ...................................................... 123, 124, 133
V
Viscardi M. ....................................................................... 134
182
W
Wrooska N. .............................................................. 146, 149
Wachowska M. ................................................................ 159
Wajda-Nikiel K. ................................................................ 151
Walczak M. ...................................................................... 135
Wawruszak A. ............................................................ 15, 136
Wawrzaszek R.......................................................... 137, 138
Wawrzaszek U. ........................................................ 137, 138
Wawszczak M. ................................................................... 66
Way A. ............................................................................. 167
Wiaderkiewicz R. ............................................................. 103
Wilmowicz E. ..................................................................... 18
Witczak A......................................................... 115, 116, 139
Witczak Z. J. ..................................................................... 107
Witerska N. ...................................................................... 140
Wnuk D. ........................................................................... 141
Wojciechowski W. ............................................................. 18
Wojtoo A. ........................................................................ 142
Woźniak A. ...................................................................... 143
Wójcik K. A. ....................................................................... 77
Wójcik M. ........................................................................ 144
Wójtowicz J. .................................................................... 145
Z
Zaczyoska D. I. ................................................................. 174
Zając A. ...................................................................... 29, 147
Zaleska O. ........................................................................ 148
Zarosa K. .......................................................................... 145
Zawadzka K. ............................................................. 146, 149
Zbrojewicz E. ................................................................... 177
Zdzisioska B. .................................................................... 152
Zglejc K. ........................................................................... 150
Zielioska M. ..................................................................... 170
Ziembik Z. .......................................................................... 48
Zuba-Surma E. ................................................................... 76
Ż
Żukowska M. ................................................................... 161
Żurek A. ............................................................... 33, 42, 152
Żyła A. .............................................................................. 122
Żyła D. ...................................................................... 108, 151
183

BOOK OF ABSTRACT

Transcription

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