Volumetric properties of aqueons solutions od 1,2-dinethylimidazolium chloride at different temperatures : bachelor thesis

U ovom radu korištenjem preciznog mjerača gustoće Anton-Paar DMA 4500M izmjerene su gustoće vodenih otopina 1,2-dimetilimidazolijevog klorida, MImCl u temperaturnom području (5 – 30 ° C) s korakom od 5 ° C u području koncentracija od ∼ 0,005 mol dm –3 do ∼ 0,1 mol dm –3 . Dobiveni eksperimentalni podaci korišteni su za izračun volumetrijskih parametara MImCl u vodi, kao što su prividni i parcijalni molarni volumeni. Korištenjem Massonove jednadžbe određeni su prividni granični molarni volumen i interakcijski koeficijent za proučavani sustav (MImCl + voda). Određena je prividna granična molarna ekspanzibilnost za MImCl u vodi pri svim radnim temperaturama – ti su podaci dali uvid u svojstva MImCl u vodi.An Anton-Paar DMA 4500M densimeter was used to measure the density for aqueous solutions of 1,2- dimethylimidazolium chloride (MImCl) in the temperature range (5 – 30 ° C) in steps of 5 ° C covering the concentration range of investigated ionic liquid ( ∼ 0,005 mol dm –3 do ∼ 0,1 mol dm –3 ). The experimental data were used to calculate the apparent molar volumes and the partial molar volumes. The volumetric data have been analyzed using Masson's equation. The limiting apparent molar volume or partial molar volume at infinite dilution, and the slope of Masson's equation at different temperatures for MImCl in water have been interpreted in terms of ion-ion and ion-solvent interactions, respectively. In addition, the limiting apparent molar expansibility value indicate structure making properties of MImCl in water

Role of protease SPRTN in DNA repair after damage caused by covalent binding of proteins

DPC-ovi predstavljaju jedan od najčešćih, ali i najmanje istraženih tipova oštećenja DNA. Njihovo istraživanje se intenziviralo tek otkrićem proteaza ovisnih o DNA, zaduženih za njihov popravak, wss1 i SPRTN. Dokazano je da se DPC-ovi popravljaju trima mehanizmima: direktnom hidrolizom kovalentne veze, izrezivanjem oštećenog dijela DNA te proteolitičkom degradacijom proteinskog dijela DPC lezije, čija međuovisnost nije poznata. U ovoj disertaciji predstavljen je razvoj novog eseja za direktnu izolaciju DPC-ova, nazvanog STAR esej, koji uspješno izolira DPC-ove različitog porijekla, pri fiziološki relevantnim uvjetima, a pri tome je dovoljno osjetljiv da omogućava vizualizaciju peptida kovalentno vezanih na DNA nastalih proteolitičkom degradacijom. Primjenom eseja utvrdili smo korake u mehanizmu popravka DPC-ova, koji se prvo proteolitički degradiraju djelovanjem SPRTN-a, prilikom čega na DNA ostaju kovalentno vezani peptidi. Peptidi se potom uklanjaju drugim mehanizmima popravka. Pokazana dinamika popravka korelira sa signalizacijom oštećenja DNA preko ATM/H2Ax osi, što je novoopisana uloga za oba proteina i neovisna je o lomovima DNA. γH2Ax je ključan za vezanje faktora popravka DNA. Ključan dokaz da je proteolitička degradacija prvi korak popravka DPC-ova proizlazi iz nemogućnosti aktivacije ovog signalnog puta u izostanku proteolize te je pokazano da DPC-ovi mogu inhibirati aktivaciju signalizacije lomova DNA lanaca ukoliko se nalaze u blizini mjesta loma. STAR esej pokazao se iznimno korisnim alatom za proučavanje dinamike nastanka i popravka DPC-ova čime će omogućiti napredak u njihovu .proučavanju.DPCs represent one of the most common and least-researched types of DNA damage. Their research intensified only after the discovery of the DNA-dependent proteases responsible for their repair, namely wss1 in yeast and SPRTN in mammals. It has been proven that DPCs are repaired by three mechanisms: direct hydrolysis of the covalent bond, excision of the damaged part of DNA, and proteolytic degradation of the protein part of the DPC lesion. However, the interdependence of these mechanisms is unknown. This dissertation presents the development of a new assay for the direct isolation of DPCs, called the STAR assay, which successfully isolates DPCs of different origins, under physiologically relevant conditions, while being sensitive enough to enable the visualization of peptides covalently bound to DNA resulting from proteolytic degradation. Using the assay, we determined the steps in the repair mechanism of DPCs, which are first proteolytically degraded by the action of SPRTN, during which covalently bound peptides remain on the DNA. Peptides are then removed by other repair mechanisms. The demonstrated dynamics of repair correlates with DNA damage signaling through the ATM/H2Ax axis, which is a newly described role for both proteins and is independent of DNA breaks. γH2Ax is crucial for binding DNA repair factors. The key evidence that proteolytic degradation is the first step in the repair of DPCs comes from the lack of activation of this signaling pathway in the absence of proteolysis, and it has been shown that DPCs can inhibit the activation of DNA strand break signaling if they are located near the break site. The STAR essay proved to be an extremely useful tool for studying the dynamics of the formation and repair of DPCs, which will enable progress in their study

Determination of farnesol and nerolidol activity on p38 MAPK cell signaling pathway : diploma thesis

Koristeći se western blot tehnikom kao metodom za identifikaciju i kvantifikaciju proteina određivan je utjecaj farnezola i nerolidola na ekspresiju p38 MAP kinaza kod tri stanične linije raka: raka mokraćnog mjehura(T24, TCC SUP) i glioblastoma (LN229). p38 MAP kinaze ključni su dio p38 MAPK staničnog signalnog puta koji je zadužen za transkripciju, popravak oštećenja na stanici i borbu protiv kancerogeneze. Na sve tri stanične linije ispitivan je utjecaj seskviterpenskih alkohola (farnezola i nerolidola) na ekspresiju p38 MAP kinaza te je rezultat uspoređen s kontrolnim uzorcima čime je ostvarena objektivna usporedba aktivnosti ispitivanih spojeva. Iz dobivenih eksperimentalnih podataka može se zaključiti da konstitucijski izomeri farnezol i nerolidol različito utječu na ispitivani signalni put u različitim staničnim linijama. Farnezol pokazuje slaba inhibitorska svojstva za T24 i TCC SUP staničnu liniju dok kod LN229 pokazuje snažnu aktivaciju signalnog puta. Nerolidol pokazuje snažnu inhibiciju kod T24 i LN229 stanične linije dok kod linije TCC SUP pokazuje aktivacijsku sposobnost. Dobiveni rezultati sugeriraju na različito vezivanje farnezola i nerolidola na stanične receptore kod istih i različitih staničnih linija što je veoma zanimljiv rezultat za daljnja istraživanja. Detaljan opis eksperimentalnog rada prikazan je u radu zajedno s rezultatima eksperimenta.Using the Western blot technique as a method for identification and quantification of proteins p38 MAP kinaze quantity was determined in three cancer cell lines: urinary bladder cancer (T24, TCC SUP) and glioblastoma (LN229). p38 MAPKs are key molecules in the p38 MAPK cell signaling pathway which is in charge of transcription, damage repairs and anti-cancer activity. All cell lines were tested for sesquiterpene alcohol (farnesol and nerolidol) activity on p38 MAPK quantity and the results was compared with control samples to gain an objective conclusion on their activity. Experimental data shows that constitutional isomers farnesol and nerolidol show different effect on tested pathway in different cell lines. Farnesol shows low inhibitory activity for T24 and TCC SUP but it shows a high activation ability for LN229 cell line. Nerolidol shows a high inhibitory activity for T24 and LN229 but it shows activation for TCC SUP cell line. Experimental data suggest that farnesol and nerolidol bind differently on the cell receptors within the same cell line and on the different cell lines making the results of this thesis interesting topic for future research. Detailed description of the experiment is shown in the thesis as well as experimental results data

Role of protease SPRTN in DNA repair after damage caused by covalent binding of proteins

DPC-ovi predstavljaju jedan od najčešćih, ali i najmanje istraženih tipova oštećenja DNA. Njihovo istraživanje se intenziviralo tek otkrićem proteaza ovisnih o DNA, zaduženih za njihov popravak, wss1 i SPRTN. Dokazano je da se DPC-ovi popravljaju trima mehanizmima: direktnom hidrolizom kovalentne veze, izrezivanjem oštećenog dijela DNA te proteolitičkom degradacijom proteinskog dijela DPC lezije, čija međuovisnost nije poznata. U ovoj disertaciji predstavljen je razvoj novog eseja za direktnu izolaciju DPC-ova, nazvanog STAR esej, koji uspješno izolira DPC-ove različitog porijekla, pri fiziološki relevantnim uvjetima, a pri tome je dovoljno osjetljiv da omogućava vizualizaciju peptida kovalentno vezanih na DNA nastalih proteolitičkom degradacijom. Primjenom eseja utvrdili smo korake u mehanizmu popravka DPC-ova, koji se prvo proteolitički degradiraju djelovanjem SPRTN-a, prilikom čega na DNA ostaju kovalentno vezani peptidi. Peptidi se potom uklanjaju drugim mehanizmima popravka. Pokazana dinamika popravka korelira sa signalizacijom oštećenja DNA preko ATM/H2Ax osi, što je novoopisana uloga za oba proteina i neovisna je o lomovima DNA. γH2Ax je ključan za vezanje faktora popravka DNA. Ključan dokaz da je proteolitička degradacija prvi korak popravka DPC-ova proizlazi iz nemogućnosti aktivacije ovog signalnog puta u izostanku proteolize te je pokazano da DPC-ovi mogu inhibirati aktivaciju signalizacije lomova DNA lanaca ukoliko se nalaze u blizini mjesta loma. STAR esej pokazao se iznimno korisnim alatom za proučavanje dinamike nastanka i popravka DPC-ova čime će omogućiti napredak u njihovu .proučavanju.DPCs represent one of the most common and least-researched types of DNA damage. Their research intensified only after the discovery of the DNA-dependent proteases responsible for their repair, namely wss1 in yeast and SPRTN in mammals. It has been proven that DPCs are repaired by three mechanisms: direct hydrolysis of the covalent bond, excision of the damaged part of DNA, and proteolytic degradation of the protein part of the DPC lesion. However, the interdependence of these mechanisms is unknown. This dissertation presents the development of a new assay for the direct isolation of DPCs, called the STAR assay, which successfully isolates DPCs of different origins, under physiologically relevant conditions, while being sensitive enough to enable the visualization of peptides covalently bound to DNA resulting from proteolytic degradation. Using the assay, we determined the steps in the repair mechanism of DPCs, which are first proteolytically degraded by the action of SPRTN, during which covalently bound peptides remain on the DNA. Peptides are then removed by other repair mechanisms. The demonstrated dynamics of repair correlates with DNA damage signaling through the ATM/H2Ax axis, which is a newly described role for both proteins and is independent of DNA breaks. γH2Ax is crucial for binding DNA repair factors. The key evidence that proteolytic degradation is the first step in the repair of DPCs comes from the lack of activation of this signaling pathway in the absence of proteolysis, and it has been shown that DPCs can inhibit the activation of DNA strand break signaling if they are located near the break site. The STAR essay proved to be an extremely useful tool for studying the dynamics of the formation and repair of DPCs, which will enable progress in their study

Role of protease SPRTN in DNA repair after damage caused by covalent binding of proteins

DPC-ovi predstavljaju jedan od najčešćih, ali i najmanje istraženih tipova oštećenja DNA. Njihovo istraživanje se intenziviralo tek otkrićem proteaza ovisnih o DNA, zaduženih za njihov popravak, wss1 i SPRTN. Dokazano je da se DPC-ovi popravljaju trima mehanizmima: direktnom hidrolizom kovalentne veze, izrezivanjem oštećenog dijela DNA te proteolitičkom degradacijom proteinskog dijela DPC lezije, čija međuovisnost nije poznata. U ovoj disertaciji predstavljen je razvoj novog eseja za direktnu izolaciju DPC-ova, nazvanog STAR esej, koji uspješno izolira DPC-ove različitog porijekla, pri fiziološki relevantnim uvjetima, a pri tome je dovoljno osjetljiv da omogućava vizualizaciju peptida kovalentno vezanih na DNA nastalih proteolitičkom degradacijom. Primjenom eseja utvrdili smo korake u mehanizmu popravka DPC-ova, koji se prvo proteolitički degradiraju djelovanjem SPRTN-a, prilikom čega na DNA ostaju kovalentno vezani peptidi. Peptidi se potom uklanjaju drugim mehanizmima popravka. Pokazana dinamika popravka korelira sa signalizacijom oštećenja DNA preko ATM/H2Ax osi, što je novoopisana uloga za oba proteina i neovisna je o lomovima DNA. γH2Ax je ključan za vezanje faktora popravka DNA. Ključan dokaz da je proteolitička degradacija prvi korak popravka DPC-ova proizlazi iz nemogućnosti aktivacije ovog signalnog puta u izostanku proteolize te je pokazano da DPC-ovi mogu inhibirati aktivaciju signalizacije lomova DNA lanaca ukoliko se nalaze u blizini mjesta loma. STAR esej pokazao se iznimno korisnim alatom za proučavanje dinamike nastanka i popravka DPC-ova čime će omogućiti napredak u njihovu .proučavanju.DPCs represent one of the most common and least-researched types of DNA damage. Their research intensified only after the discovery of the DNA-dependent proteases responsible for their repair, namely wss1 in yeast and SPRTN in mammals. It has been proven that DPCs are repaired by three mechanisms: direct hydrolysis of the covalent bond, excision of the damaged part of DNA, and proteolytic degradation of the protein part of the DPC lesion. However, the interdependence of these mechanisms is unknown. This dissertation presents the development of a new assay for the direct isolation of DPCs, called the STAR assay, which successfully isolates DPCs of different origins, under physiologically relevant conditions, while being sensitive enough to enable the visualization of peptides covalently bound to DNA resulting from proteolytic degradation. Using the assay, we determined the steps in the repair mechanism of DPCs, which are first proteolytically degraded by the action of SPRTN, during which covalently bound peptides remain on the DNA. Peptides are then removed by other repair mechanisms. The demonstrated dynamics of repair correlates with DNA damage signaling through the ATM/H2Ax axis, which is a newly described role for both proteins and is independent of DNA breaks. γH2Ax is crucial for binding DNA repair factors. The key evidence that proteolytic degradation is the first step in the repair of DPCs comes from the lack of activation of this signaling pathway in the absence of proteolysis, and it has been shown that DPCs can inhibit the activation of DNA strand break signaling if they are located near the break site. The STAR essay proved to be an extremely useful tool for studying the dynamics of the formation and repair of DPCs, which will enable progress in their study

Seasonality of Polyaromatic Hydrocarbons (PAHs) and Their Derivatives in PM_2.5 from Ljubljana, Combustion Aerosol Source Apportionment, and Cytotoxicity of Selected Nitrated Polyaromatic Hydrocarbons (NPAHs)

Airborne particulate matter (PM) is a vector of many toxic pollutants, including polyaromatic hydrocarbons (PAHs) and their derivatives. Especially harmful is the fine fraction (PM2.5), which penetrates deep into the lungs during inhalation and causes various diseases. Amongst PM2.5 components with toxic potential are nitrated PAHs (NPAHs), knowledge of which is still rudimentary. Three of the measured NPAHs (1-nitropyrene (1-nP), 9-nitroanthracene (9-nA), and 6-nitrochrysene (6-nC)) were detected in ambient PM2.5 from Ljubljana, Slovenia, along with thirteen non-nitrated PAHs. The highest concentrations of pollutants, which are closely linked with incomplete combustion, were observed in the cold part of the year, whereas the concentrations of NPAHs were roughly an order of magnitude lower than those of PAHs throughout the year. Further on, we have evaluated the toxicity of four NPAHs, including 6-nitrobenzo[a]pyrene (6-nBaP), to the human kidney cell line, HEK293T. The most potent was 1-nP (IC50 = 28.7 µM), followed by the other three NPAHs, whose IC50 was above 400 or 800 µM. According to our cytotoxicity assessment, atmospheric 1-nP is the most harmful NPAH among the investigated ones. Despite low airborne concentrations of NPAHs in ambient air, they are generally considered harmful to human health. Therefore, systematic toxicological assessment of NPAHs at different trophic levels, starting with cytotoxicity testing, is necessary in order to accurately evaluate their threat and adopt appropriate abatement strategies

Chemical Composition and Biological Activity of <i>Salvia officinalis</i> L. Essential Oil

In our study, we investigated the chemical composition and cytotoxic activity of essential oils isolated from Dalmatian sage (Salvia officinalis L.) collected along the Adriatic coast of Croatia. Scanning electron microscopy (SEM) was used to examine the morphology of the stem and leaf surfaces. Essential oil excretory glands were detected on both the leaves and stem surfaces. The essential oils were isolated by hydrodistillation, and their chemical composition was determined by gas chromatography and mass spectrometry (GC-MS). Sage essential oils were mixtures of terpene compounds, among which the most common were: α- and β-thujone, camphor, and 1,8-cineol. Cytotoxic activity was tested using MTS assay on multiple cell lines: normal and immortalized fibroblasts (HF77FA and HDF-Tert), immortalized lung line (BEAS-2B), and breast adenocarcinoma (MDA-MB-231). The growth of treated cells was determined relative to control conditions without treatment. The immortalized lung line was the least resistant to the activity of the essential oils, whereas immortalized fibroblasts were the most resistant. Statistical analysis has connected the cytotoxic effect and chemical composition of the studied essential oils. To the best of our knowledge, this work is the first testing of the cytotoxic activity of S. officinalis EO’s on the BEAS-2B, HF77FA, and HDF-Tert cell lines. The presented data on essential oil chemical composition and cytotoxic effect on 4 types of human cells supports pharmacotherapeutic potential this plant is known to have

Centaurea triumfetii essential oil chemical composition, comparative analysis, and antimicrobial activity of selected compounds

Abstract The essential oils from the Centaurea genus are well known for their pharmacological properties. The most abundant and dominant chemical components in Centaurea essential oils are ß-caryophyllene, hexadecanoic acid, spathulenol, pentacosane, caryophyllene oxide, and phytol. However, whether these dominant components are the key drivers for observed antimicrobial activity remains unclear. Thus, the aim of this study was dual. Here we provide comprehensive, literature-based data to correlate the chemical compounds in Centaurea essential oils with the tested antimicrobial activity. Secondly, we characterized the essential oil of Centaurea triumfettii All. squarrose knapweed using coupled system gas chromatography–mass spectrometry and tested its phytochemicals for antimicrobial activity against E. coli and S. epidermis using disc diffusion assay and monitoring their growth in Muller Hinton broth. The most abundant compounds in C. triumfettii essential oil were hexadecanoic acid (11.1%), spathulenol (10.8%), longifolene (8.8%), germacrene D (8.4%), aromadendrene oxide (6.0%) and linoleic acid (5.3%). Based on our analysis of literature data from other Centaurea essential oils, they were positively correlated with antimicrobial activity. Using an agar disk diffusion method, tested chemical constituents did not show experimental evidence to support this positive correlation to antimicrobial activity when we tested them as pure components. The antibacterial effect of essential oil constituents may be related to a complex synergistic, rather than a single component as suggested by performed network pharmacology analysis, underlying the theoretical interactions between the essential oil phytochemicals listed as potentially responsible for antimicrobial activity and should be confirmed in further in-depth studies. This is the first report on the comparative analysis of Centaurea essential oils with good antimicrobial activity, as well as the first analysis of chemical components of the essential oil from C. triumfettii and the first report of antimicrobial activity of the representative, pure components: aromadendrene, germacrene D, spathulenol, longifolene, and the mixture of selected chemical compounds. This work contributes to the body of knowledge on the genus Centaurea and C. triumfettii species

The Essential Oil Composition of Helichrysum italicum (Roth) G. Don: Influence of Steam, Hydro and Microwave-Assisted Distillation

Helichrysum italicum (Roth) G. Don (Asteraceae), also known as immortelle, usually grows in the Mediterranean area. The composition of the essential oil (EO) of immortelle is a mixture of various aromatic substances, mainly monoterpenes and sesquiterpenes. Distillation is the most widely used method for extraction of EO immortelle, although the yield is very low (&lt;1%). In this work, we aim to investigate how the use of different distillation methods affects the yield and chemical composition of immortelle EO. For this purpose, we applied two conventional methods: steam distillation (SD) and hydrodistillation (HD), and a modern (environmentally friendly) technique&mdash;microwave-assisted distillation (MAD). Wild immortelles from four different locations in Croatia were collected and carefully prepared for extraction. Each sample was then analyzed by gas chromatography&ndash;mass spectrometry (GC-MS). GraphPad Prisma statistical software was used to study the statistics between different groups of connections and analyze the data on the number of connections. The results show that HD gives a significantly higher yield (0.31 &plusmn; 0.09%) compared to MAD (0.15 &plusmn; 0.03%) and SD (0.12 &plusmn; 0.04%). On the other hand, the highest number of chemical compounds was identified with MAD (95.75 &plusmn; 15.31%), and most of them are subordinate compounds with complex structures. SD isolated EOs are rich in derived acyclic compounds with the highest percentage of ketones. The results show that the application of different distillation methods significantly affects the composition of the obtained immortelle EO, considering the yield of EO, the number of isolated, derived and non-derived compounds, chemotypes and compounds with simple (acyclic) and complex structures