Histology and Embryology Students' Science Association
HESA Studenckie Koło Naukowe
przy Katedrze i Zakładzie Histologii i Embriologii WUM
Osiągnięcia w roku akademickim 2012/13
Projekty przyznane do realizacji w roku akademickim 2012/2013:
1M15/NM5/12 Charakterystyka śmierci komórki w wyniku przebiegu procesu entozy - kontynuacja projektu badawczego - kierownik projektu Agnieszka Sarnecka,
opiekun projektu: dr n. med. Łukasz Biały
1M15/NM3/12 Badanie indukowalności i stopnia nasilenia procesu apoptozy na liniach ludzkich białaczek poddanych działaniu inhibitorów proteasomów kierownik projektu: Jarosław Bartkowski i Mateusz Kęcik,
opiekun projektu dr n. med Izabela M-Biały
1M15/NM4/12 Cytostatyczne/ cytotoksyczne właściwości nowych pochodnych kwasu galusowego. kierownik projektu: Sebastian Kowalski,
opiekun projektu dr n. med Marek Kujawa
1M15/NM2/12 Aktywacja wybranych kinaz należących do szlaku mTOR w brodawczaku i rakusplotu naczyniówkowego u dzieci. kierownik projektu: Beata Rak,
opiekun projektu dr n. med Jarosław Jóźwiak
1M15/NM8/12 Guzy medulloblastoma u dzieci- badanie udziału kinaz Akt i Erk w rozwojunowotworu. kierownik projektu: Anna Borowska,
opiekun projektu dr n. med Jarosław Jóźwiak
UDZIAŁ STUDENTÓW W KONFERECJACH KRAJOWYCH:
III Konferencja Naukowa Studenckich Kół Naukowych przy Centrum Biostruktury "Postępy w badaniach podstawowych" Warszawa 17 listopada 2012
1. Anna Grzyb, mgr Aneta Federowicz Rola wariantów allelicznych w sekwencjach niekodujących genów
2. Magdalena Bałkowiec Rola mTOR w chorobie Parkinsona
3. lek. med. Stanisław Szlufik Rola mTOR w chorobie Alzheimera
4. Anna Borowska Aktywacja szlaków Akt/PKB oraz MAPK/Erk w rdzeniakach
5. Anna Kula Złośliwy nowotwór osłonek nerwów obwodowych
6. Paulina Karolczak, Katarzyna Sułek Pochodne kwasu galusowego i kumaryny jako potencjalne leki przeciwnowotworowe
7. Barbara Szladowska Techniczne aspekty wykonywania metody badawczej Western blot.
8. Hubert Kołodziejski Ocena reaktywności prawidłowej błony maziowej na czynniki zewnętrzne
9. Agnieszka Sarnecka, Urszula Szewczak Zjawisko komórki w komórce – porównanie kanibalizmu, emperipolezy i entozy
10. Sylwia Gajda Badanie zjawiska entozy na linii komórek raka trzustki.
Streszczenia prac studenckich prezentowanych podczas 9th WIMC maj 2013:
Session: Endocrinology and Diabetology [213]
Treatment of TSH-secreting human pituitary adenomas: comparison of octreotide and pasireotide in vitro
Marcin Miadziołko, Radosław Napora, Sebastian Kowalski
Trustees of the paper: Emir Sajjad, MD, prof. Grzegorz Zieliński, MD, PhD, Izabela Młynarczuk-Biała, MD, PhD
Introduction: The strategy of treatment in pituitary adenomas (PAs) depends to a great extent on the hormonal activity. Somatostatin analogs inhibit tumor secretion and growth, through interaction with somatostatin receptors (SSTR). Each somatostain analogue has aspecific level of affinity to different types of SSTRs. Octreotide (OCT), act primarily by binding to somatostatin receptor 2 (SSTR2). Recently the universal or “pan-receptor” somatostatin ligand pasireotide (SOM230) has been developed, which possess high affinity binding to SSTR2, 3, 5 and moderate affinity for SSTR 1. Its use has been approved for the treatment of ACTH secreting adenomas.
Aim of the study: In our study we compare the in vitro effect of both somatostain analogs on TSH secreting pituitary adenomas (TSHomas). TSHomas account for less than 2% of PAs and often pose a clinical challenge.
Material and methods: TSHomas were excised transsphenoidaly from two male patients (KF: invasive, recurrent, poor response to somatostain analogs/JP: non invasive, first surgery). Both had similar SSTR expression pattern. Fresh adenoma tissue was dispersed and cultured. Confluent primary cell cultures were established and supplemented with OCT and SOM230 in four concentrations (0.1 nM, 1 nM, 10 nM, 100 nM). TSH levels were measured in culture medium after 24, 48 and 72 h. Caspase-3 activity was assessed as an indicator of cell apoptosis after 48 h using flow cytometry in wells containing 1 nM, 10 nM and 100 nM of each agent. The cells were also pictured using confocal microscopy.
Results: In KF paradoxical elevation of hormone levels in all setting raging from 185% to 670% was observed. JP showed inhibitory effect on hormone secretion with both agents. As expected the hormone levels where the lowest after 72 h with SOM230 inhibiting the hormone secretion at a higher level comparing to OCT (at 1 nM 54% vs. 76%, 10 nM 53% vs. 76% of the control hormone level). Both adenomas showed no increase of caspase-3activity or apoptotic morphology in confocal microscopy comparing to controls.
Session: Genetics & Molecular Biology [239]
Identification of a a novel HLXB9 mutation in patient with Currarino syndrome
Marta Żeberkiewicz, Maciej Baranowski,Radosław Maksym
Trustee of the paper: Radosław Maksym, MD
Introduction: Currarino syndrome is a rare congenital disorder described in 1981 as the association of three main features: sacral malformation (sickle-shaped sacrum or sacral agenesis), hindgut abnomaly and presacral mass. Mutation of homeotic HLXB9 gene, located at 7q36, is disease-causing. Affected gene encodes a transcription factor that regulates early embryological development through a highly conserved homeodomain. Thus far about 50 different heterozygous mutations of HLXB9 have been reported in patients with Currarino syndrome.
Aim of the study: The aim of the study was to describe the molecular background and confirm clinical diagnosis of Currarino syndrome in 32-year-old woman with incidentally diagnosed presacral teratoma and sacral malformation. The presence of mutation was investigated in proband’s family members.
Material and methods: Genomic DNA was isolated from peripheral blood of patients by salt precipitation. Coding sequence and intron/exon boundarieof HLXB9 gene was amplified by PCR with specific primers. Sequence was obtained by direct bidirectional sequencing of amplicones.
Results: HLXB9 sequencing revealed a heterozygotus transversion c800C>A that resulted in truncation of peptide (Ser267X). Detected mutation have been never described in literature before. The same mutation was found in asymptomatic father of proband. This nonsense mutation affects highly conserved homeodomain region that is involved in the protein-DNA binding. No other mutations within the HLXB9 gene were identified in the family.
Conclusions: Autosomal dominant transmission connected with variable expression and incomplete penetrance creates obstacles in clinical diagnosis of Currarino syndrome. Moreover, some symptoms of the disorder can be present in other syndromes (e.g. Hirsprumg disease). Clinical diagnosis in patients presenting sacral agenesis, anorectal malformation, constipation, anterior meningocoele, lipoma/teratoma, malignancy, perianal sepsis and spinal cord tethering should be confirm by molecular diagnosis. The technique implemented in by Student Research Group of The Department of Histology and Embryology allows reliable and simple confirmation of clinical diagnosis of Currarino syndrome by molecular genetic analysis.
Session: Genetics & Molecular Biology [247]
Development of a method for identification of RHO gene mutations in patients with retinitis pigmentosa
Rafał Sejdak, Marta Podgorskarsejdak@gmail.com
Trustee of the paper: dr n. med. Monika Ołdak
Introduction: Retinitis pigmentosa (RP) is the most frequent hereditary disease of the eye leading to blindness. Mutations in more than 60 different genes may cause RP. One of these genes is RHO encoding rhodopsin. Mutations in RHO are responsible for approximately 30% of the autosomal dominant (AD) form of RP.
Aim of the study: In this study a method for identification of RHO gene mutations has beendescribed.Material and methods: PCR primers were designed and PCR conditions were optimized for amplification of the entire RHO coding region. Next, PCR products were sequenced using the modified Sanger method. The study was conducted in a group of 78 Polish patients (63 families) with RP, including 22 families with AD RP.
Results: Four different previously reported heterozygous RHO gene mutations were found in four families. In families with AD RP three non-synonymous mutations p.Thr17Met, p.Arg135Trp, p.Pro171Leu were identified. They account for RP in 13.6% (3/22) of the investigated families with AD RP. One synonymous mutation was identified in a patient with a negative RP family history.
Conclusions: The study characterizes patients for mutations in the RHO gene and shows that the frequency of RHO gene mutations in Polish patients with AD RP is lower than in previously investigated populations.
3rd award Session: Genetics & Molecular Biology [248]
The molecular diagnosis of Fuchs corneal dystrophy in Polish population
Ewelina Ruszkowska, Aneta Federowicz
ewelina.ruszkowska19@gmail.com
Trustee of the paper: dr n. med. Monika Ołdak
Introduction: The TCF4 gene on chromosome 18q encodes the E2-2 protein, a transcription factor involved in cellular growth and differentiation. Recent studies have revealed a strong association of the TCF4 single nucleotide polymorphism (rs613872) in intron 3 with late-onset FCD.
Aim of the study: The purpose of this study was to investigate the association of the rs613872 with FCD in Polish patients.
Material and methods: Patients with symptomatic FCD (n = 67; 53 females and 14 males, mean age: 68} 14.6) and age-matched controls (n = 251) were enrolled in the study. The diagnosis of FCD phenotype was performed by visualization of “guttea” and stromal oedema with slit-lamp examination and by in vivo confocal microscopy (CS3, Nidek Technologies). Total genomic DNA was isolated from blood and rs613872 was genotyped in both groups using TagMan SNP genotyping assay. Allele and genotype frequencies were compared between the groups by Chi square test. Odds ratio (OR) with 95% confidence intervals (95% CI), assuming dominant model, were calculated to estimate risk.
Results: Distribution of the TT, TG and GG genotypes were 72.5%, 24.7% and 2.8% in control subjects and 25.4%, 62.7% and 11.9% in FCD patients, respectively. The frequency of allele T and G in the patient group was 76/134 (56.7%) and 58/134 (43.3%). In the control group the respective allele frequencies were 426/502 (84.8%) and 76/502 (15.2%). Allel G was significantly more prevalent in patients with FCD than among control subjects (OR = 7.76, 95%CI: 4.19-14.37, χ2 = 49.4, p < 0.0001).
Conclusions: In the present study, polymorphic variant of TCF4 (rs613872) showed strong association with FCD, verifying its significant role as a disease susceptibility gene also in Polish patients.
1st award Session: Pharmacy [472]
Examination of cytostatic/cytotoxic effects induced by novel Cu(II) metal complex of new derivates of coumarin
Sebastian Kowalski, Agnieszka Czajkowska, Julia Borowska
Medical University of Warsaw
Trustee of the paper: Izabela Młynarczuk-Biały,MD, PhD
Introduction: Coumarin (1,2-benzopyrone) and their derivates are important and useful compounds with diverse pharmacological properties. They have a wide range of applications as antitumor, anti-HIV, anticoagulant, antibacterial, antioxidant and antiinflammatory agents. A large number of structurally novel coumarin derivates have ultimately been reported to show substantial cytotoxic activity in vitro. Investigations have proved that binding of drug (i.e. ligand) to a metalloelement enhances its activity and complexes possess even more cytostatic properties than the parental ligands.
Aim of the study: The aim of the present work is evaluation of biological activity of fifteen new coumarin derivatives with copper Cu(II).
Material and methods: Series of new coumarin derivates and their complexes with metal ions were designed and synthesized in the Department of Organic Chemistry at Faculty of Pharmacy, Medical University of Warsaw. To examine potential antitumor activity the novel compounds were tested on following cell lines: Du145 (prostate cancer), HL60 (human myeloid leukemia) and NIH3T3 (mouse fibroblasts). As a screening test cells were incubated with the new compounds at concentrations of 100 and 10 μM for 48 hours. Cytostatic activity was evaluated by performing resazurin based PrestoBlue viability test. The most promising compounds were tested further to determine the optimal concentration range. At that stage the activity of the complexes and parent ligands were compared: cells were incubated for 48 in 96-well plates using a range of concentrations (500, 300, 200, 100, 50, 20 and 10 μM). Cell cycle analysis was performed by flow cytometry – cells were incubated with complexes (48 hours), then collected and processed. To assess the apoptosis of the cells the annexin V test and propidium iodide staining followed by flow cytometry was performed.
Results: We established a methodology that allowed to select three compounds with the greatest impact on cancer cell lines, and no negative effects on non tumorogenic NIH3T3 cells line. The optimum concentration range of 50-300 μM was established and followed by cell cycle analysis and apoptosis evaluation.
Conclusions: Our study has shown that the copper complexes of the new coumarin derivatives induce apoptosis in greater extent than the parent ligands and are active against tumor cells.