Primena masene spektrometrije u oblasti funkcionalne proteomike na primeru Arion vulgaris i Drosophila melanogaster

Functional proteomics represent an approach which could provide answers to two major questions: the revelation of biological functions of unknown proteins and the description of cellular mechanisms at the protein level. The Spanish slug, Arion vulgaris, is one of the hundred most invasive species in Central Europe. No transcriptomic or proteomic study on A. vulgaris has been carried out so far. In this thesis, the first transcriptomic database from adult specimens of A. vulgaris is reported. To facilitate and enable proteomics in this non-model organism, a mRNA-derived protein database was constructed for protein identification. A gel-based proteomic approach was used to obtain the first generation of a comprehensive slug mantle proteome. A total of 2128 proteins were unambiguously identified from which 48 proteins represent novel proteins with no significant homology in NCBI non-redundant database. Combined transcriptomic and proteomic analysis revealed an extensive repertoire of novel proteins with a role in innate immunity, including many associated pattern recognition proteins, effector proteins and cytokine-like proteins. The immense and very successful adaptation and spreading of A. vulgaris suggests that it developed highly effective mechanisms to deal with infections and natural predators. The number and diversity in gene families encoding lectins point to a complex defense system, probably as a result of adaptation to a pathogen-rich environment. These results are providing a fundamental and important resource for subsequent studies on molluscs, as well as for putative antimicrobial compounds for drug discovery and biomedical applications...Funkcionalna proteomika danas predstavlja metodu koja može da ukaže na odgovor na dva pitanja: koja je biološka funkcija novootkrivenih proteina i da okarakteriše ćelijske procese na proteinskom nivou. Arion vulgaris predstavlja jednu od sto najinvaznivijih vrsta u Evropi. Do sada u nauĉnoj literaturi nisu zabeleženi podaci o iRNK i proteinskom profilu ove vrste. U prvom delu teze obraĊena je data problematika gde su prikazani rezultati analize iRNA na osnovu ĉega je napravljena baza podataka koja je korišćena za identifikaciju proteina. Ukupan broj identifikovanih proteina, dobijenih tripsinskom digestijom u gelu i analizom na masenom spektrometru, iznosi 2128. Od ukupnog broja indentifikovanih proteina, 48 nema homologe u nacionalnom centru za biotehnološke informacije (NCBI) i predstavljaju do sada neopisane proteine. Kombinacijom transkriptoma i proteoma opisan je ĉitav repertoar kako novih, tako i već poznatih proteina koji imaju ulogu u uroĊenom imunom sistemu, kao na primer „associated pattern recognition“, efektorski i citokinu-sliĉni proteini. Dobijeni rezultati ukazuju na mehanizam borbe protiv infekcija i prirodnih neprijatelja, koji verovatno omogućavaju uspešnu adaptaciju i široku rasprostranjenost ove vrste. Velika raznovrsnost gena koji kodiraju lektine ukazuje na odbrambeni sistem vrste Arion vulgaris u sredini koji je bogata patogenima. Rezultati koji su proizašli iz ove teze pružaju fundametalno znanje o proteomskom profilu ove vrste mekušaca, ukljuĉujući i 48 novih proteina. Drugi deo teze je fokusiran na „gel-free bottom-up“ analizu proteina iz membranskih frakcija glave Drosophila melanogaster..

Environmental Exposure to Metals, Parameters of Oxidative Stress in Blood and Prostate Cancer: Results from Two Cohorts

We studied the potential role of exposure to various metal(oid)s (As, Cd, Cr, Hg, Ni, and Pb) in prostate cancer. Two cohorts were established: the Croatian cohort, consisting of 62 cases and 30 controls, and the Serbian cohort, consisting of 41 cases and 61 controls. Blood/serum samples were collected. Levels of investigated metal(oid)s, various parameters of oxidative stress, and prostate-specific antigen (PSA) were determined in collected samples. A comparison of the measured parameters between 103 prostate cancer patients and 91 control men from both Croatian and Serbian cohorts showed significantly higher blood Hg, SOD, and GPx levels and significantly lower serum SH levels in prostate cancer patients than in controls. Correlation analyses revealed the significant relationship between certain parameters of oxidative stress and the concentrations of the measured metal(loid)s, pointing to the possible role of metal(oid)-induced oxidative stress imbalance. Furthermore, a significant inverse relationship was found between the blood Pb and the serum PSA in prostate cancer patients, but when the model was adjusted for the impacts of remaining parameters, no significant association between the serum PSA and the measured parameters was found. The results of the overall study indicate a substantial contribution of the measured metal(loid)s to the imbalance of the oxidant/antioxidant system. Although somewhat conflicting, the results of the present study point to the possible role of investigated metal(oid)s in prostate cancer, especially for Hg, since the obtained relationship was observed for both cohorts, followed by the disturbances in oxidative stress status, which were found to be correlated with Hg levels. Nevertheless, further studies in larger cohorts are warranted to explain and confirm the obtained results

Mikrocistin-LR u površinskim vodama reke Ponjavice

Background/Aim. Cyanobacterial toxins befall a group of various compounds according to chemical structure and health effects on people and animals. The most significant in this large group of compounds are microcystins. Their presence in water used for human consumption causes serious health problems, liver beeing the target organ. Microcystins are spread all over the world. Waterblooms of cyanobacterias and their cyanotoxins are also common in the majority of surface waters in Serbia. The aim of this study was to propose HPLC method for determination of mikrocystin-LR, to validate the method and to use it for determination of microcystin-LR in the surface water of the river Ponjavica. The Ponjavica is very eutrophic water and has ideal conditions for the cyanobacterial growth. Methods. Sample of water form the Ponjavica river were collected during the summer 2008. Coupled columns (HLB, Sep-Pak), were used for sample preparation and HPLC/PDA method was used for quantification of microcystin- LR. Results. Parameters of validation show that the proposed method is simple, fast, sensitive (0.1 mg/L) and selective with the yield of 89%-92%. The measuring uncertainty of 5% was obtained. The obtained results for surface water show that microcystin concentration reached the maximum level during August and September (1.5 μg/L). The value is higher than maximum allowable concentration of microcystin in drinking water (1 μg/L) proposed by WHO. Conclusion. This study contributes to the issue of pollution of the National Park Ponjavica. Besides, literature data and WHO clearly point out harmfulness of cyanobasterias and their toxins and implicate the necessity of legislation concerning determination and monitoring of these toxins in our country. Method used for quentification of mycrocystin-LR was shown to be sensitive, selective, rapid and simple and could be recommended for routine determination of this toxin.Uvod/Cilj. Cijanotoksini, odnosno toksini cijanobakterija, spadaju u grupu raznovrsnih jedinjenja kako po hemijskoj strukturi, tako i po dejstvu na ljude i životinje. Najveći značaj u ovoj velikoj grupi jedinjenja imaju mikrocistini. Njihovo prisustvo u vodi može da izazove toksične efekte, posebno na jetru. Mikrocistini su rasprostranjeni širom sveta. Cvetanje toksičnih cijanobakterija i njihovi cijanotoksini karakteristični su i za neke površinske vode u Srbiji. Cilj ovog rada bio je da se uvede HPLC metoda za određivanje mikrocistina-LR, da se izvrši validacija metode i odredi sadržaj mikrocistina-LR u površinskoj vodi reke Ponjavice, veoma eutrofne reke, koja poseduje idealne uslove za razvoj cijanobakterija. Metode. Za pripremu uzoraka korišćeni su HLB i C8 ulošci, a za dodatno prečišćavanje upotrebljeni su Sep-Pak silika ulošci. Kvantifikacija mikrocistina-LR vršena je metodom HPLC pomoću detektora na principu fotodiode (HPLC/PDA) korišćenjem kolone RP C18 Zorbax Eclips (4,6x150,5 μm), upotrebom mobilne faze sastava 0,1% rastvor trifluorosirćetne kiseline (TFA) u metanolu: 0,1% vodeni rastvor TFA u odnosu 60 : 40 (v/v), na 238 nm. Retenciono vreme mikrocistina-LR iznosilo je 10 min. Uzorci vode reke Ponjavice za analizu mikrocistina-LR sakupljeni su tokom perioda jun-novembar 2008. godine. Rezultati. Validacijom metode utvrđena je osetljivost 0,1 μg/L, prinos 89-92% i merna nesigurnost od ± 5%. Rezultati uzoraka vode reke Ponjavice pokazali su da je vrednost mikrocistina-LR dostigla maksimum tokom avgusta i septembra (1,5 μg/L), i da je iznad maksimalno dozvoljene koncentracije za pijaću vodu (1 μg/L) prema preporuci Svetske zdravstvene organizacije. Zaključak. Parametri validacije pokazuju da je predložena metoda osetljiva i selektivna, što je, uz činjenicu da je jednostavna i brza, čini pogodnom za rutinsko određivanje mikrocistina-LR. Predloženom metodom određene su povišene koncentracije ovog toksina cijanobakterija u uzorcima vode reke Ponjavice, koje ukazuju na zagađenost Parka prirode 'Ponjavica'

Kinetics of dsb induction and changes in cell cycle regulation in melanoma cells after ionizing radiation

The effects of γ-rays on the DNA level, i.e. formation of double-strand breaks and expression of p21 were studied in vitro on the human HTB 140 melanoma cells. Cells were exposed to the dose range from 2 to 16 Gy. Effects were analyzed 30 min, 2, 6 and 24 h after irradiation. It has been shown that the level of phosphorylated histone H2AX (γH2AX) is time- and dose-dependent, as well as the expression of p21.Physical chemistry 2012 : 11th international conference on fundamental and applied aspects of physical chemistry; Belgrade (Serbia); 24-28 September 201

Sensitivity of lung carcinoma cells to y-rays and erlotinib

In order to increase radio-sensitivity of human lung adenocarcinoma NCI-H1568 cells, targeted therapy drug, erlotinib was used. The impact of radiation and erlotinib on cell behaviour was analyzed using three biological endpoints. Irradiations with γ-rays resulted in reduction of cell survival, viability and proliferation. Erlotinib significantly inhibited cell growth and proliferation capacity. Combined treatments with radiation and erlotinib showed high level of reduction of cell viability and proliferation. Preliminary data encourage further investigations of mechanisms underlying the radiation responses enhanced by erlotinib.Physical chemistry 2012 : 11th international conference on fundamental and applied aspects of physical chemistry; Belgrade (Serbia); 24-28 September 201

Radiation dose determines the method for quantification of DNA double strand breaks

Ionizing radiation induces DNA double strand breaks (DSBs) that trigger phosphorylation of the histone protein H2AX (gamma H2AX). Immunofluorescent staining visualizes formation of gamma H2AX foci, allowing their quantification. This method, as opposed to Western blot assay and Flow cytometry, provides more accurate analysis, by showing exact position and intensity of fluorescent signal in each single cell. In practice there are problems in quantification of gamma H2AX. This paper is based on two issues: the determination of which technique should be applied concerning the radiation dose, and how to analyze fluorescent microscopy images obtained by different microscopes. HTB140 melanoma cells were exposed to gamma-rays, in the dose range from 1 to 16 Gy. Radiation effects on the DNA level were analyzed at different time intervals after irradiation by Western blot analysis and immunofluorescence microscopy. Immunochemically stained cells were visualized with two types of microscopes: AxioVision (Zeiss, Germany) microscope, comprising an ApoTome software, and AxioImagerA1 microscope (Zeiss, Germany). Obtained results show that the level of gamma H2AX is time and dose dependent. Immunofluorescence microscopy provided better detection of DSBs for lower irradiation doses, while Western blot analysis was more reliable for higher irradiation doses. AxioVision microscope containing ApoTome software was more suitable for the detection of gamma H2AX foci

Radiosensitization of Non-Small Cell Lung Carcinoma By Egfr Inhibition

Molecular targeted cancer therapy is a promising treatment strategy. Considering the central role of the epidermal growth factor receptor in cell proliferation and survival, there are indications that targeted agents like tyrosine kinase inhibitors, i. e., erlotinib, may enhance the antitumor treatment by radiation. The aim of this study is to analyze the inactivation effects of gamma-rays and to test the radiosensitizing potential of erlotinib on human lung adenocarcinoma cells in vitro. Irradiations were performed with doses ranging from 1 Gy to 8 Gy. In order to increase the radiosensitivity of CRL-5876 lung adenocarcinoma cells, the cells were treated with a clinically relevant concentration of 2 mu M erlotinib. The effects of single and combined treatments were monitored using clonogenic survival, cell viability and proliferation assays at different time points. For the detection and visualization of the phosphorylated histone H2AX (gamma-H2AX), an important biological marker of DNA double-strand break formation, fluorescence inununocytochemistry, was performed. The response to the treatment was monitored at four time points: 30 min, 2, 6, and 24 h. Irradiations with gamma-rays resulted in significant cell inactivation regarding all analyzed biological endpoints. Combined treatments revealed consistent cell inactivation. Moreover, compared to gamma-rays alone, elevated levels of gamma-H2AX foci were observed after pretreatment with erlotinib, indicating radiosensitization through impaired DNA repair

Cancer Incidence in a Population Living Near a Petrochemical Facility and Oil Refinery

There is growing concern that pollution from petrochemical and oil refinery installations in Pan~evo (Serbia) has increased the incidence of various diseases including cancer. The aim of our study was to investigate cancer incidence in Pan~evo and to compare it with the region of central Serbia. Cancer incidence data were obtained from the corresponding Serbian Cancer Registries. Systematic local monitoring of benzene, toluene and methyl mercaptane in the atmosphere within Pan~evo’s industrial area indicated that the average monthly and yearly concentrations often exceeded permitted levels proposed by EU standards (5 mgm–3). Cancer incidence was lower in Pan~evo than in central Serbia (the standardised incidence for all types of cancers in Pan~evo was 218.3 and in central Serbia it was 241.7 per 100,000 inhabitants). The available data do not allow us to correlate air pollution in Pan~evo with increased cancer incidence

Myeloid Cells Restrict MCMV and Drive Stress-Induced Extramedullary Hematopoiesis through STAT1

Cytomegalovirus (CMV) has a high prevalence worldwide, is often fatal for immunocompromised patients, and causes bone marrow suppression. Deficiency of signal transducer and activator of transcription 1 (STAT1) results in severely impaired antiviral immunity. We have used cell-type restricted deletion of Stat1 to determine the importance of myeloid cell activity for the defense against murine CMV (MCMV). We show that myeloid STAT1 limits MCMV burden and infection-associated pathology in the spleen but does not affect ultimate clearance of infection. Unexpectedly, we found an essential role of myeloid STAT1 in the induction of extramedullary hematopoiesis (EMH). The EMH-promoting function of STAT1 was not restricted to MCMV infection but was also observed during CpG oligodeoxynucleotide-induced sterile inflammation. Collectively, we provide genetic evidence that signaling through STAT1 in myeloid cells is required to restrict MCMV at early time points post-infection and to induce compensatory hematopoiesis in the spleen