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Namen: Ugotoviti, ali lahko z aplikacijo visokih odmerkov vitamina C zmanjšamo tveganje za pljučni edem po porodu pri bolnicah s težko preeklampsijo. Hipotezi: Vitamin C v prvih treh dneh po porodu pri bolnicah s težko preeklampsijo zmanjša količino zunajžilne pljučne tekočine merjeno z ultrazvočno (UZ) preiskavo pljuč. Vitamin C v prvih treh dneh po porodu pri bolnicah s težko preeklampsijo zmanjša koncentracijo biokemičnih označevalcev oksidativnega stresa v urinu. Utemeljitev: Preeklampsija je vodilni vzrok za pojav obporodnega pljučnega edema. Pomemben vzrok za to je okvarjena endotelna funkcija in posledično povečana žilna prepustnost. Eden od možnih vzrokov za endotelno disfunkcijo pri preeklampsiji je tudi povečan oksidativni stres. Antioksidant vitamin C pri življenjsko ogroženih bolnikih dokazano zmanjša žilno prepustnost. Vpliv vitamina C na potek težke preeklampsije po porodu ni raziskan. Metode: Monocentrična, randomizirana, s placebom kontrolirana, dvojno slepa študija. Štiriintrideset bolnic s težko preeklampsijo je prejelo intravensko vitamin C (1,5g/6h) (n=17) ali placebo (n=17) prve tri dni po porodu. UZ pljuč je bil narejen enkrat dnevno, od prvega do tretjega dne po porodu. Količino zunajžilne pljučne tekočine smo opredelili z ECS (echo comet score seštevek števila B-linij v 28-interkostalnih oknih). V urinu odvzetem prvi in tretji dan po porodu so bile določene vrednosti štirih označevalcev oksidativnega stresa: ditirozin, 8-hidroksi-2-deoksigvanozin (8- OHdG), 8-izoprostan in N-epsilon-Heksanoil-Lizin (HEL). Uporabljen je bil Mann-Whitney U-test za statistično primerjavo med skupinama. Vrednost p ? 0,05 je bila upoštevana za statistično pomembno. Preučevani izidi: Pri dan po porodu nismo našli statistično pomembne razlike v ECS med skupinama (mediana 23 (inter-kvartilni razmik (IQR) 21–61) vs 18 (IQR 8–35) p = 0,31). Drugi in tretji dan po porodu je bil ECS statistično pomembno manjši v skupini, ki je prejemala vitamin C, v primerjavi s placebom (8 (IQR 3–14) vs 35 (IQR 15–78) p = 0,03 in 5 (IQR 3–10) vs 18 (IQR 18– 44) p = 0,04). Vrednosti ditirozina in 8-OHdG se prvi dan po porodu med skupinama niso statistično pomembno razlikovale (p = 0,23 in p = 0,77). Bile pa so pomembno manjše tretji dan po porodu v skupini, ki je prejemala vitamin C (p = 0,04 in p = 0,03). Vrednosti 8-izoprostana in HEL se niso statistično pomembno razlikovale ne prvi (p = 0,41 in p = 0,42), ne tretji (p = 0,25 in p = 0,24) dan po porodu. Zaključek: Visoki intravenski odmerki vitamina C pri bolnicah s težko preeklampsijo niso zmanjšali količine zunajžilne pljučne tekočine ali markerjev oksidativnega stresa v urinu prvi dan po porodu. Vplivali pa so na zmanjšanje zunajžilne pljučne tekočine drugi in tretji dan po porodu ter na zmanjšanje nivojev ditirozina in 8-OHdG (markerjev oksidativne poškodbe proteinov in DNA) tretji dan po porodu. Objective: To determine if vitamin C in high doses reduces extravascular lung water in women with severe preeclampsia postpartum. Hypotheses: Vitamin C in the first three days postpartum reduces extravascular lung water assessed by lung ultrasound in patients with severe preeclampsia. Vitamin C in the first three days postpartum reduces urinary levels of oxidative stress biomarkers. Rationale: Preeclampsia is the leading cause of peripartum pulmonary edema which occurs, at least in part, due to endothelial dysfunction and increased vascular permeability. Oxidative stress contributes significantly to the development of preeclampsia and its clinical course. Vitamin C was proven to be beneficial in reducing vascular permeability in critically ill patients. It has been shown that the use of vitamin C in pregnancy was not effective for primary or secondary prevention of preeclampsia. The effect of vitamin C on preeclampsia postpartum as a tertiary prevention measure, i.e., reducing risks of complications once preeclampsia has already occurred, is not known. Population: Consecutively admitted patients with singleton pregnancies complicated by severe preeclampsia. Methods: Single-center, randomized, placebo-controlled, double-blind trial. Thirty-four patients received vitamin C (1.5 g/6 hours) (n = 17) or placebo (n = 17) intravenously at days 1, 2, and 3 postpartum. A lung ultrasound was performed once daily in the first three days following delivery. Echo Comet Score (ECS) was obtained using the 28-rib interspaces technique. Urine was collected on postpartum days 1 and 3. It was analysed for four biomarkers of oxidative stress: dityrosine, 8-hydroxy-2-deoxyguanosine (8- OHdG), 8-isoprostane, and N-epsilon-Hexanoyl-Lysine (HEL). Mann-Whitney-U test was used to compare vitamin C vs placebo groups. A p 䁤 0.05 was considered statistically significant. Main outcome measures: ECS on day 1 postpartum was the primary outcome studied. ECS on days 2 and 3 postpartum as well as urinary biomarkers of oxidative stress (dityrosine, 8- OHdG, 8-isoprostane, and HEL, normalized for urinary creatinine), on days 1 and 3 postpartum were analyzed as secondary outcomes. Results: There was no significant difference in ECS on day 1 (median 23 (inter-quartile range (IQR) 21–61) vs 18 (IQR 8–35) p = 0.31). On days 2 and 3, ECS was significantly lower in the vitamin C group compared to the placebo (8 (IQR 3–14) vs 35 (IQR 15–78) p = 0.03 and 5 (IQR 3–10) vs 18 (IQR 18– 44) p = 0.04, respectively). Dityrosine and 8-OHdG values did not differ significantly between the two study groups on day 1 after delivery (p = 0.23 and p = 0.77, respectively), but were significantly lower in the vitamin C group compared to the placebo group on day 3 after delivery (p = 0.04 and p = 0.03, respectively). Values of 8-isoprostane and HEL did not differ significantly between the two study groups on day 1 (p = 0.41 and p = 0.42, respectively), as well as on day 3 after delivery (p = 0.25 and p = 0.24, respectively). Conclusion: High dose of intravenous vitamin C did not reduce the amount of extravascular lung water or decrease markers of oxidative stress in postpartum patients with severe preeclampsia on day 1 after delivery. It seems, however, it has a delayed effect with a reduction in extravascular lung water detected on ultrasound on days 2 and 3 following delivery and a reduction of urinary levels of dityrosine and 8-OHdG (markers of protein and DNA oxidative damage, respectively) on day 3 after delivery.

Rak trebušne slinavke je kljub napredku v znanosti in medicini ena od rakavih bolezni z najslabšo napovedjo, saj je ob postavitvi diagnoze le petina bolnikov primernih za kirurško zdravljenje, medtem ko je pri večini bolezen že v napredovalih fazah oz. je razsejana. Bolezen zaradi poznega pojava simptomov in njihove nespecifičnosti predstavlja zahteven diagnostični izziv, zaradi česar se vse več raziskav usmerja v nove načine zdravljenja in spremljanja bolnikov, ki prejemajo terapijo. Trenutno so v ta namen v rutinski uporabi predvsem različne slikovne preiskave in določanje tumorskih označevalcev, medtem ko na podlagi dognanj različnih študij, ki so dokazale da so visoke vrednosti reaktivnih kisikovih spojin, spremembe v redoks zaporedjih in slabo uravnavanje redoks signaliziranja pogosti kazalniki napredovanja raka in odpornosti na terapijo, vse več pozornosti posvečajo označevalcem oksidativnega stresa. Preučevanje njihovih koncentracij v urinu predstavlja učinkovit in neinvaziven način ugotavljanja, kako se redoks ravnovesje spreminja na daljši rok, saj se z urinom izločajo oksidirani produkti, nastali ob popravljanju poškodovane DNK. V magistrski nalogi smo se na podlagi izsledkov predhodnih raziskav odločili preveriti, kakšne so možnosti uporabe določenih označevalcev oksidativnega stresa v urinu pri pacientih z napredovalim rakom trebušne slinavke. Zanje smo se odločili zato, ker naj bi predstavljali trenutno najbolj priporočljive oziroma obetavne označevalce za oceno oksidativnega statusa na daljši rok. Izbrali smo naslednje analite: 8-hidroksideoksigvanozin, 8-izoprostan, ditirozin in heksanoil lizin, ki predstavljajo produkte oksidativnih poškodb nukleinskih kislin, proteinov ter lipidov. Meritev koncentracij izbranih analitov smo opravili s pomočjo komercialno dostopnih testnih kompletov ELISA. Rezultate smo primerjali glede na trenutno najbolj široko uporabnima tumorskima označevalcema CA 19-9 in CEA ter stopnjo napredovanja bolezni in učinkovitost terapije, pri preiskovancih. Z analizo naših rezultatov smo pri preiskovancih z rakom trebušne slinavke, glede na dostopne referenčne vrednosti, zaznali nekatere trajne oz. značilno spremenjene koncentracije analitov. Tako smo ugotovili, da se napredovanje bolezni pogosto kaže kot naraščanje koncentracij 8-hidroksideoksigvanozina, 8-izoprostana, ditirozina in heksanoil lizina, medtem ko učinkovita terapija koncentracije znižuje. Z upoštevanjem podatkov o stadiju bolezni smo prav tako dokazali, da nižje vrednosti 8-hidroksideoksigvanozina ob prvem odvzemu vzorca urina pogosto napovedujejo boljše preživetje. Po drugi strani pa smo ob primerjavi s tumorskima označevalcema CA 19-9 in CEA ugotovili, da vrednosti preučevanih analitov ne korelirajo vedno z njunimi nivoji, pri čemer je visoko stopnjo korelacij izkazoval le ditirozin. Na podlagi izsledkov naše raziskave menimo, da bi bilo označevalce oksidativnega stresa v urinu smiselno vpeljati v algoritem spremljanja bolnikov z napredovalim rakom trebušne slinavke. Pancreatic cancer is, despite advances in science and medicine, one of the cancer diseases with the worst prognosis. Only one fifth of all patients are suitable for surgical treatment upon diagnosis, while the majority are already in advanced stages of the disease with metastases present. Due to the late onset of symptoms and their non-specificity, the disease represents an arduous diagnostic challenge, which is why more and more research is being directed towards new ways of treating and monitoring patients who are receiving therapy. Currently, various imaging tests and measurement of tumor markers are in routine use for this purpose. However, based on the findings of various studies, which proved that high levels of reactive oxygen compounds, changes in redox sequences and poor regulation of redox signalling are common indicators of cancer progression and resistance to therapy, more and more attention is being paid to markers of oxidative stress. Examining their concentrations in urine is an efficient and non-invasive way of determining how the redox balance has changed in the long term, as oxidized products that were formed during the repair of damaged DNA are excreted in the urine. Based on previous research, this assignment aims to check the possibilities of using certain markers of oxidative stress in urine of patients with advanced pancreatic cancer. We decided for those because they are currently the most recommended or promising markers for long-term assessment of oxidative status. We selected the analytes 8-hydroxydeoxyguanosine, 8-isoprostane, dityrosine and hexanoyl lysine, which represent the products of oxidative damage to nucleic acids, proteins and lipids. The concentrations of selected analytes were measured using commercially available ELISA test kits. The results were compared to currently most widely used tumour markers CA 19-9 and CEA, as well as regarding the degree of disease progression and the effectiveness of the therapy received by the subjects. By analysing the data obtained during the research, we detected some permanent and characteristically changed concentrations of analytes in subjects with pancreatic cancer compared to available reference values. We found that the progression of the disease is often manifested as an increase in concentration of 8-hydroxydeoxyguanosine, 8-isoprostane, dityrosine and hexanoyl lysine, while effective therapy lowers concentrations. Based on data on various disease stages, we also proved that lower values of 8-hydroxydeoxyguanosine at the first sampling often predict a better survival. On the other hand, when compared to the tumour markers CA 19-9 and CEA, we found that the values of 8-hydroxydeoxyguanosine, 8-isoprostane, dityrosine and hexanoyl lysine do not always correlate with them, and only dityrosine showed a high proportion of correlations. Based on the results of the our research, it would be reasonable to introduce markers of oxidative stress in the urine into the monitoring algorithm of patients with advanced pancreatic cancer.

Rtanjski čaj, Satureja montana L. ima široku upotrebu u mnogim delovima sveta, koristi se kao kulinarska biljka i u tradicionalnoj medicini za lečenje ili ublažavanje različitih zdravstvenih simptoma. Međutim, brojne studije su pokazale da biljka ima farmakološki potencijal da se upotrebljava i kao antibakterijski, antidijabetesni, anti-HIV i antiproliferativni lek. Farmakološki efekti rtanjskog čaja mogu se pripisati prisustvu fitohemikalija poput flavonoida, sterola, tanina i etarskih ulja. Fenolna jedinjenja su uglavnom odgovorna za antioksidativnu aktivnost biljke Satureja montana L. U ovom istraživanju cilj je bio utvrđivanje uticaja ekstrakta biljke Satureja montana L. na parametre kao što su glikemija, status lipida i biohemijski parametri, zatim na indikatore koji ukazuju na funkciju jetre i bubrega u serumu zdravih, dijabetičnih pacova i pacova koji su bili na ishrani obogaćenoj mastima, kao i de se ispita uticaj na pokazatelje oksidativnog stresa kod životinja izloženih toksičnoj dozi paracetamola. Za određivanje hipoglikemijskog i antihiperlipidemijskog dejstva biljke Satureja montana L., laboratorijski pacovi soja WISTAR su podeljeni u 8 grupa, u grupama koje su korišćene za određivanje hipoglikemijskog potencijala dijabetes je indukovan streptozotocino nakon čega su životinje per os 28 dana, tretirane fiziološkim rastvorom, metforminom 100 mg/kg, ekstraktom rtanjskog čaja 500 mg/kg i njihovom kombinacijom. Za ispitivanje antihiperlipidemijskog efekta životinje su bile na ishrani obogaćenoj mastima i fiziološkim rastvorom, simvastatinom 10 mg/kg, ekstraktom rtanjskog čaja 500 mg/kg i njihovom kombinacijom. Za određivanje antioksidativnog potencijala biljke Satureja montana L. životinje su podeljene u četiri grupe i 7 dana su tretirane ekstraktom rtanjskog čaja i fiziološkim rastvorom. Nakon toga, pacovi su bili izloženi toksičnoj dozi paracetamola. U životinjskom serumu određivani su biohemijski parametri, koncentracija lipida i parametri funkcije bubrega i jetre. Određivanje aktivnosti lipidne peroksidacije i enzima antioksidativne zaštite u homogenatima jetre eksperimentalnih životinja, nakon primene toksične doze paracetamola, je ispitivano ex vivo. In vitro ispitivanja su vršena sa ekstraktima dobijenim rastvaranjem u različitim rastvaračima. Ispitivanja antioksidativne aktivnosti, kao i ukupni nivoi fenola i flavonoida, određeni su spektrofotometrijskom metodom. Kod životinja sa indukovanim dijabetesom 28-dnevni tretman ekstraktom Satureja montana L. u poređenju sa kontrolom grupom dijabetičnih životinja prouzrokovao je statistički značajno smanjenje glukoze. Na kraju studije, u grupi koja je primala ekstrakt Satureja montana L., uočene su statistički značajno niže vrednosti triglicerida u odnosu na kontrolnu dijabetičnu grupu. Na druge parametre lipidnog statusa, ispitivani ekstrakt nije pokazao značajne efekte. Kod životinja čiju smo ishranu obogatili mastima primenom ekstrakta Satureja montana L. došlo je do statistički značajnog snižavanja LDL holesterola u serumu, u odnosu na kontrolnu grupu koja je bila na isharni obogaćenoj mastima. Vrednosti parametara pokazatelja funkcije bubrega i jetre: AST, urea, kreatinin i mokraćna kiselina bile su značajno niže u grupi tretiranoj ekstraktom Satureja montana L. u odnosu na kontrolnu grupu koja je bila na ishrani obogaćenoj mastima. Pretretman životinja biljnim ekstraktom pre primene toksične doze paracetamola značajno je smanjio aktivnost alkalne fosfataze, koncentraciju direktnog bilirubina i kreatinina u serumu u odnosu na grupu tretiranu fiziološkim rastvorom i pojedinačnom dozom paracetamola. Aktivnost lipidne peroksidaze bila je značajno veća kod životinja koje su tretirane fiziološkim rastvorom u poređenju sa tretmanom ekstraktom Satureja montana L., pre toksične doza paracetamola. Pored toga, smanjen je broj inflamatornih ćelija zajedno sa aktivnošću enzima CIP2E1 kod životinja koje su prethodno tretirane ekstraktom Satureja montana L. Rezultati ovih istraživanja pokazali su hipoglikemijski potencijal ekstrakta Satureja montana L. kod životinja sa indukovanim dijabetesom. Pretretman ekstraktom Satureja montana L. pre primene toksične doze paracetamola, pokazao je potencijal hepatoprotektivnog, antiinflamatornog i antioksidativnog dejstva. Takođe, PCA analiza je pokazala da veliki uticaj na antioksidativni efekat imaju medijumi korišćeni za ekstrakciju., Satureja montana L. is widely used as culinary herb and in traditional medicine for the treatment or relief of various common health symptoms in many parts of the world. However, numerous studies have shown that it has pharmacological potential to be used as antibacterial drug, antidiabetic, anti-HIV and antiproliferative drug. Its pharmacological effects can be attributed to the presence of phytochemicals like flavonoids, sterols, tannins and essential oils. Phenolic compounds are mostly responsible for the anti-oxidative activity of Satureja montana L. In this research, the aim was to determine the influence of Satureja montana L. extract on glycemia, lipid status and biochemical parameters, indicators of liver and kidney function in the serum of healthy and diabetic rats and rats fed with high fat diet, as well as to examine it influence on the parameters of oxidative stress in animals exposed to the toxic dose of paracetamol. For determination of hypoglycemic and antihyperlipidemic effect of Satureja montana L., Wistar laboratory rats were divided into 8 groups, in groups used to determine hypoglycemic potential diabetes was induced by streptozotocin and after that, animals were treated for 28 days per os with saline solution, metformin 100 mg/kg, Satureja montana extract 500 mg/kg and their combination. For examination of hypolipidemic effect animals were treated with high fat diet and saline solution, simvastatin 10 mg/kg, Satureja montana L. extract 500 mg/kg and their combination. For determination of antioxidant potential of Satureja montana L. animals were divided into four groups and winter savory extract and saline solution were given them orally for 7 days. After that, rats were exposed to the toxic dose of paracetamol. Biochemical parameters, the lipids concentration and parameters of the kidney and liver function, were determined in animal serum. Ex vivo analyses included the determination of lipid peroxidation activity and the activity of antioxidant protection enzymes in liver homogenates of the test animals, after administration of a toxic dose of paracetamol. In vitro analyses were performed with extracts obtained with different solvents. Antioxidant activity tests as well as total phenolic and flavonoid content, were determined spectrophotometrically. In diabetic animals, 28 days treatment with Satureja montana L. extract brought to statistically significant decrease of glucose comparing to the control. Statistically significantly lower TG values were observed in the group that received Satureja montana L. extract compared to the control diabetic group, at the end of the study. On the other parameter of lipid status, the examined extract did not show significance effects. In animals fed with food enriched with fats, application of Satureja montana L. extract brought to statistically significant decrease of LDL cholesterol, relative to the control group fed with food enriched with fats. Concentration of parameters of the kidney and liver function: AST, urea, creatinine, and uric acid were significantly lower in the group treated with Satureja montana L. extract in comparison to the control group fed with food enriched with fats. Pre-treatment of animals with herbal extracts before application of the toxic dose of paracetamol significantly decreased alkaline phosphatase activity, concentration of direct bilirubin and creatinine in the serum relative to the group treated with saline solution and single dose of paracetamol. The activity of lipid peroxidation was significantly higher in animals that were treated with saline solution compared with Satureja montana L. extract, before the toxic doses of paracetamol. In addition, in animals pretreated with Satureja montana L. extract, the number of inflammatory cells is decreased along with the activity of CYP2E1 enzymes. The results of these studies showed hypoglycemic potential of Satureja montana L. extract in animals with induced diabetes. Pretreatment with Satureja montana L. extract prior to the administration of the toxic dose of paracetamol has shown potential of hepatoprotective, anti-inflammatory and antioxidative effect. Also, PCA analyses showed that the extraction mediums had a large influence on the antioxidative effect.

Rtanjski čaj, Satureja montana L. ima široku upotrebu u mnogim delovima sveta, koristi se kao kulinarska biljka i u tradicionalnoj medicini za lečenje ili ublažavanje različitih zdravstvenih simptoma. Međutim, brojne studije su pokazale da biljka ima farmakološki potencijal da se upotrebljava i kao antibakterijski, antidijabetesni, anti-HIV i antiproliferativni lek. Farmakološki efekti rtanjskog čaja mogu se pripisati prisustvu fitohemikalija poput flavonoida, sterola, tanina i etarskih ulja. Fenolna jedinjenja su uglavnom odgovorna za antioksidativnu aktivnost biljke Satureja montana L. U ovom istraživanju cilj je bio utvrđivanje uticaja ekstrakta biljke Satureja montana L. na parametre kao što su glikemija, status lipida i biohemijski parametri, zatim na indikatore koji ukazuju na funkciju jetre i bubrega u serumu zdravih, dijabetičnih pacova i pacova koji su bili na ishrani obogaćenoj mastima, kao i de se ispita uticaj na pokazatelje oksidativnog stresa kod životinja izloženih toksičnoj dozi paracetamola. Za određivanje hipoglikemijskog i antihiperlipidemijskog dejstva biljke Satureja montana L., laboratorijski pacovi soja WISTAR su podeljeni u 8 grupa, u grupama koje su korišćene za određivanje hipoglikemijskog potencijala dijabetes je indukovan streptozotocino nakon čega su životinje per os 28 dana, tretirane fiziološkim rastvorom, metforminom 100 mg/kg, ekstraktom rtanjskog čaja 500 mg/kg i njihovom kombinacijom. Za ispitivanje antihiperlipidemijskog efekta životinje su bile na ishrani obogaćenoj mastima i fiziološkim rastvorom, simvastatinom 10 mg/kg, ekstraktom rtanjskog čaja 500 mg/kg i njihovom kombinacijom. Za određivanje antioksidativnog potencijala biljke Satureja montana L. životinje su podeljene u četiri grupe i 7 dana su tretirane ekstraktom rtanjskog čaja i fiziološkim rastvorom. Nakon toga, pacovi su bili izloženi toksičnoj dozi paracetamola. U životinjskom serumu određivani su biohemijski parametri, koncentracija lipida i parametri funkcije bubrega i jetre. Određivanje aktivnosti lipidne peroksidacije i enzima antioksidativne zaštite u homogenatima jetre eksperimentalnih životinja, nakon primene toksične doze paracetamola, je ispitivano ex vivo. In vitro ispitivanja su vršena sa ekstraktima dobijenim rastvaranjem u različitim rastvaračima. Ispitivanja antioksidativne aktivnosti, kao i ukupni nivoi fenola i flavonoida, određeni su spektrofotometrijskom metodom. Kod životinja sa indukovanim dijabetesom 28-dnevni tretman ekstraktom Satureja montana L. u poređenju sa kontrolom grupom dijabetičnih životinja prouzrokovao je statistički značajno smanjenje glukoze. Na kraju studije, u grupi koja je primala ekstrakt Satureja montana L., uočene su statistički značajno niže vrednosti triglicerida u odnosu na kontrolnu dijabetičnu grupu. Na druge parametre lipidnog statusa, ispitivani ekstrakt nije pokazao značajne efekte. Kod životinja čiju smo ishranu obogatili mastima primenom ekstrakta Satureja montana L. došlo je do statistički značajnog snižavanja LDL holesterola u serumu, u odnosu na kontrolnu grupu koja je bila na isharni obogaćenoj mastima. Vrednosti parametara pokazatelja funkcije bubrega i jetre: AST, urea, kreatinin i mokraćna kiselina bile su značajno niže u grupi tretiranoj ekstraktom Satureja montana L. u odnosu na kontrolnu grupu koja je bila na ishrani obogaćenoj mastima. Pretretman životinja biljnim ekstraktom pre primene toksične doze paracetamola značajno je smanjio aktivnost alkalne fosfataze, koncentraciju direktnog bilirubina i kreatinina u serumu u odnosu na grupu tretiranu fiziološkim rastvorom i pojedinačnom dozom paracetamola. Aktivnost lipidne peroksidaze bila je značajno veća kod životinja koje su tretirane fiziološkim rastvorom u poređenju sa tretmanom ekstraktom Satureja montana L., pre toksične doza paracetamola. Pored toga, smanjen je broj inflamatornih ćelija zajedno sa aktivnošću enzima CIP2E1 kod životinja koje su prethodno tretirane ekstraktom Satureja montana L. Rezultati ovih istraživanja pokazali su hipoglikemijski potencijal ekstrakta Satureja montana L. kod životinja sa indukovanim dijabetesom. Pretretman ekstraktom Satureja montana L. pre primene toksične doze paracetamola, pokazao je potencijal hepatoprotektivnog, antiinflamatornog i antioksidativnog dejstva. Takođe, PCA analiza je pokazala da veliki uticaj na antioksidativni efekat imaju medijumi korišćeni za ekstrakciju. Satureja montana L. is widely used as culinary herb and in traditional medicine for the treatment or relief of various common health symptoms in many parts of the world. However, numerous studies have shown that it has pharmacological potential to be used as antibacterial drug, antidiabetic, anti-HIV and antiproliferative drug. Its pharmacological effects can be attributed to the presence of phytochemicals like flavonoids, sterols, tannins and essential oils. Phenolic compounds are mostly responsible for the anti-oxidative activity of Satureja montana L. In this research, the aim was to determine the influence of Satureja montana L. extract on glycemia, lipid status and biochemical parameters, indicators of liver and kidney function in the serum of healthy and diabetic rats and rats fed with high fat diet, as well as to examine it influence on the parameters of oxidative stress in animals exposed to the toxic dose of paracetamol. For determination of hypoglycemic and antihyperlipidemic effect of Satureja montana L., Wistar laboratory rats were divided into 8 groups, in groups used to determine hypoglycemic potential diabetes was induced by streptozotocin and after that, animals were treated for 28 days per os with saline solution, metformin 100 mg/kg, Satureja montana extract 500 mg/kg and their combination. For examination of hypolipidemic effect animals were treated with high fat diet and saline solution, simvastatin 10 mg/kg, Satureja montana L. extract 500 mg/kg and their combination. For determination of antioxidant potential of Satureja montana L. animals were divided into four groups and winter savory extract and saline solution were given them orally for 7 days. After that, rats were exposed to the toxic dose of paracetamol. Biochemical parameters, the lipids concentration and parameters of the kidney and liver function, were determined in animal serum. Ex vivo analyses included the determination of lipid peroxidation activity and the activity of antioxidant protection enzymes in liver homogenates of the test animals, after administration of a toxic dose of paracetamol. In vitro analyses were performed with extracts obtained with different solvents. Antioxidant activity tests as well as total phenolic and flavonoid content, were determined spectrophotometrically. In diabetic animals, 28 days treatment with Satureja montana L. extract brought to statistically significant decrease of glucose comparing to the control. Statistically significantly lower TG values were observed in the group that received Satureja montana L. extract compared to the control diabetic group, at the end of the study. On the other parameter of lipid status, the examined extract did not show significance effects. In animals fed with food enriched with fats, application of Satureja montana L. extract brought to statistically significant decrease of LDL cholesterol, relative to the control group fed with food enriched with fats. Concentration of parameters of the kidney and liver function: AST, urea, creatinine, and uric acid were significantly lower in the group treated with Satureja montana L. extract in comparison to the control group fed with food enriched with fats. Pre-treatment of animals with herbal extracts before application of the toxic dose of paracetamol significantly decreased alkaline phosphatase activity, concentration of direct bilirubin and creatinine in the serum relative to the group treated with saline solution and single dose of paracetamol. The activity of lipid peroxidation was significantly higher in animals that were treated with saline solution compared with Satureja montana L. extract, before the toxic doses of paracetamol. In addition, in animals pretreated with Satureja montana L. extract, the number of inflammatory cells is decreased along with the activity of CYP2E1 enzymes. The results of these studies showed hypoglycemic potential of Satureja montana L. extract in animals with induced diabetes. Pretreatment with Satureja montana L. extract prior to the administration of the toxic dose of paracetamol has shown potential of hepatoprotective, anti-inflammatory and antioxidative effect. Also, PCA analyses showed that the extraction mediums had a large influence on the antioxidative effect.

Aripiprazol (ARI) in olanzapin (OLA) sta pogosto uporabljena atipična antipsihotika s številnimi prednostmi pri zdravljenju shizofrenije in bipolarne motnje. Oba antipsihotika izkazujeta zaščitno delovanje, vendar so ob dolgotrajni terapiji zlasti problematične metabolne komplikacije, katerih vzroki so slabo poznani in bi jih potencialno lahko povezali z nepravilnim delovanjem jeter. Jetra so namreč osrednji organ presnove in detoksikacije obeh antipsihotikov, zato bi lahko motene celične funkcije bistveno pripomogle k razvoju opaženih učinkov. Namen dela je bil raziskati morebitne škodljive in zaščitne učinke ARI in OLA v celičnih modelih jeter. Proučili smo odziv celic na kratkotrajne in dolgotrajne tretmaje z izbranima antipsihotikoma ter vpliv zunanjega vira oksidativnega stresa. ARI je za jetrne celice bolj citotoksičen v primerjavi z OLA. OLA namreč ne izkazuje neposrednega citotoksičnega vpliva na jetrne celice niti po večtedenski izpostavitvi, medtem ko je učinek ARI takojšen, in sicer v obliki mitohondrijske hiperpolarizacije. Mitohondrijska hiperpolarizacija znatno upočasni celični metabolizem in delitev celic ter vodi v trajno povečan oksidativni stres. Prav ta pa bi lahko hkrati imel znaten prispevek k zaščitnemu delovanju ARI proti zunanjemu viru oksidativnega stresa, saj morajo celice vseskozi vzdrževati visok nivo antioksidativne obrambe. Zaščitni učinek ARI vsaj deloma poteka preko antioksidativnih encimov po dolgotrajni izpostavitvi in ni neposredno povezan z uravnavanjem apoptoze, avtofagije in proteasoma. ARI ščiti celice Fao in primarne hepatocite, medtem ko smo zaščitni učinek OLA opazili zgolj v kratkotrajnih poskusih na primarnih hepatocitih. Dolgotrajna izpostavitev celic antipsihotikoma je bistveno pripomogla k relevantnosti raziskave, saj je razkrila učinke, ki jih zgolj s kratkotrajnimi tretmaji ne bi uspeli zaznati. Predstavljen celični model kronične izpostavitve antipsihotikom torej bolje odraža delovanje učinkovin v ravnotežnem stanju in je potencialno uporaben tudi pri proučevanju drugih zdravil s predvidenimi dolgotrajnimi terapijami. Aripiprazole (ARI) and olanzapine (OLA) are commonly used atypical antipsychotics with many benefits in the treatment of schizophrenia and bipolar disorder. Despite of protective effects of both antipsychotics, there are metabolic complications, particularly during long-term therapy. The causes are poorly understood and could potentially be associated with liver dysfunction. The liver is the central organ of metabolism and detoxification of both antipsychotics. Impaired cellular functions could significantly contribute to the development of the observed effects. The purpose of this study was to investigate the potential adverse and protective effects of ARI and OLA in liver cell models. We studied the cellular response to short-term and long-term antipsychotic treatments exposed to acute oxidative stress. ARI is more cytotoxic to liver cells compared to OLA. OLA does not have a direct cytotoxic effect on liver cells even after several weeks of exposure, while the effect of ARI is immediate, in the form of mitochondrial hyperpolarization. Mitochondrial hyperpolarization significantly slows down cellular metabolism as well as cell division and leads to permanently elevated oxidative stress. The latter could be also linked with the protective role of ARI against external sources of oxidative stress, as cells must permanently maintain the high level of antioxidant defense. The protective effect of ARI is at least partially mediated by endogenous antioxidant enzymes after prolonged ARI exposure and it is not directly linked with the regulation of apoptosis, autophagy or proteasome activity. ARI protected Fao cells and primary hepatocytes, while the protective effect of OLA was observed only on primary hepatocytes during acute treatment. Long-term exposure to antipsychotics revealed effects that could not be detected with short-term treatments alone. Therefore, the proposed cellular model of chronic exposure to antipsychotics better reflects the action of drugs in the equilibrium and is also potentially useful to study the effects of other drugs used in long-term therapies.

Alcohol use disorder (AUD) is a highly prevalent, comorbid, and disabling disorder. The underlying mechanism of ethanol neurotoxicity and the involvement of oxidative stress is still not fully elucidated. However, ethanol metabolism has been associated with increased oxidative stress through alcohol dehydrogenase, the microsomal ethanol oxidation system, and catalase metabolic pathways. We searched the PubMed and genome-wide association studies (GWAS) catalog databases to review the literature systematically and summarized the findings focusing on AUD and alcohol abstinence in relation to oxidative stress. In addition, we reviewed the ClinicalTrials.gov resource of the US National Library of Medicine to identify all ongoing and completed clinical trials that include therapeutic interventions based on antioxidants. The retrieved clinical and preclinical studies show that oxidative stress impacts AUD through genetics, alcohol metabolism, inflammation, and neurodegeneration.

The coronavirus disease (COVID-19) pandemic is a leading global health and economic challenge. What defines the disease’s progression is not entirely understood, but there are strong indications that oxidative stress and the defense against reactive oxygen species are crucial players. A big influx of immune cells to the site of infection is marked by the increase in reactive oxygen and nitrogen species. Our article aims to highlight the critical role of oxidative stress in the emergence and severity of COVID-19 and, more importantly, to shed light on the underlying molecular and genetic mechanisms. We have reviewed the available literature and clinical trials to extract the relevant genetic variants within the oxidative stress pathway associated with COVID-19 and the anti-oxidative therapies currently evaluated in the clinical trials for COVID-19 treatment, in particular clinical trials on glutathione and N-acetylcysteine.

The aim of this study was to determine the effects of a specific mixture of metal nanoparticles (Ag, Cu, Fe and MnО2) orally administered to rats, on biochemical blood markers as indicators of chronic toxicity. Animals (Wistar line rats (n=20)) in experimental groups were given a solution of a mixture of metal salts or a mixture of nanoparticles with food for 90 days. On days 15, 30, 60 and 90 of the experiment, five rats from each group were anaesthetised with CO2 and decapitated, and blood samples were collected for biochemical studies and spectrophotometric measurement. The toxic effect of the chronic administration of the mixture at a dose of 4.0 mg/kg of body weight in white rats caused partial immunosuppression expressed as hypoproteinaemia, excessive formation of circulating immune complexes and acute phase serum mucoid proteins (P, Cilj je naše studije bio utvrditi učinke mješavine nanočestica određenih metala (Ag, Cu, Fe i MnО2) koje su oralno davane štakorima na biokemijske markere krvi kao indikatore kronične toksičnosti. Životinjama (štakorima soja Wistar (n=20)) je u eksperimentalnim skupinama hranom tijekom 90 dana davana je otopina mješavine metalnih soli ili mješavine nanočestica. Na 15., 60. i 90. dan eksperimenta, pet štakora je iz svake skupine pomoću CO2 anestezirano i dekapitirano te su prikupljeni uzorci krvi za biokemijske studije. Spektrofotometrijska mjerenja su provedena uporabom SHIMADZU UV-1800 instrumenta. Toksični je učinak kronične primjene NPMe mješavine pri dozi od 4,0 mg/kg tjelesne mase u bijelih štakora izazvao djelomičnu imunosupresiju izraženu kao hipoproteinemija, pretjerano formiranje cirkulirajućih imunokompleksa i mukoidne proteine u serumu (P

Sistemska skleroza (SSc) je sistemska avtoimunska revmatska bolezen, katere patogeneza ni popolnoma razjasnjena, tudi obvladovanje glavnih kliničnih znakov, kot sta vnetje in fibroza, s farmakoterapijo pogosto ni učinkovito. Z namenom boljšega razumevanja patogeneze in odkrivanja novih terapevtskih učinkovin smo proučevali oksidativni stres in migracijo monocitov pri bolnikih s SSc ter učinke adiponektina in resolvina D1 na vnetje in fibrozo. Metodološki pristop je zajemal delo na vzorcih bolnikov s SSc in zdravih oseb, celičnih kulturah in živalskem modelu. Pri bolnikih s SSc smo izmerili povišan indeks oksidativnega stresa (na osnovi povišanih serumskih reaktivnih kisikovih metabolitov in/ali antioksidantne kapacitete), več prelomov DNA v levkocitih ter oslabljene popravljalne mehanizme poškodb DNA. Na površini monocitov bolnikov s SSc smo dokazali povečano izražanje adhezijske molekule CD62L, katere izražanje je bilo spodbujeno s komponentami seruma bolnikov. Spremembe v oksidativnem stanju in migratornem fenotipu monocitov so pri bolnikih s SSc sovpadale s pljučno prizadetostjo, prisotnostjo avtoprotiteles in uporabo farmakoterapije. Na skeletnomišičnih celicah in skeletnomišičnih fibroblastih smo pokazali, da se adiponektin vpleta v vnetje in mišično regeneracijo z aktivacijo znotrajceličnih signalnih poti ter s spodbujanjem izločanja interlevkina-6 in sfingozin-1-fosfata, kot možne posledice povečane ceramidazne aktivnosti adiponektinskih receptorjev. AdipoRon, analog adiponektina, primerljivih učinkov ni izkazal. Resolvin D1 je na mišjem modelu peritonealne fibroze znižal delež polimorfonuklearnih celic v sistemskem krvnem obtoku, medtem ko vpliva na lokalno vnetje in fibrozo v trebušni steni ni imel. Negativno je vplival na izražanje hemerina, regulatorja vnetnih procesov, ki se veže na isti receptor kot resolvin E1. V doktorski disertaciji smo pokazali, kako obsežnejše stanje oksidativnega stresa in večji potencial migracije monocitov pri SSc sovpada s klinično sliko bolezni ter tako pripomogli k boljšemu razumevanju patogeneze. Obenem smo dokazali, da ima adiponektin učinke na mišično vnetje in regeneracijo, resolvin D1 pa potencial obvladovanja vnetja v fibrotičnih boleznih tudi z regulacijo izražanja ligandov resolvinskih receptorjev. Systemic sclerosis (SSc) is a systemic autoimmune rheumatic disease whose pathogenesis remains poorly understood, and pharmacotherapy for the major clinical manifestations - inflammation and fibrosis - is often ineffective. To better understand the pathogenesis and find new therapeutic options, we investigated oxidative stress and monocyte migration in SSc and effects of adiponectin and resolvin D1 on inflammation and fibrosis. Our methodological approach included work with samples from SSc patients, cell cultures, and animal models. SSc patients exhibited a higher oxidative stress index (based on increased serum reactive oxygen metabolites and/or antioxidant capacity), more DNA breaks in leukocytes, and impaired DNA damage repair mechanisms. We found increased surface expression of the adhesion molecule CD62L on SSc monocytes and showed that CD62L expression is stimulated by SSc serum components. Both the altered oxidative status and monocyte phenotype were consistent with lung involvement, autoantibodies, and pharmacotherapy in SSc patients. In skeletal muscle cells and skeletal muscle fibroblasts, adiponectin acted on inflammation and muscle regeneration by activating intracellular signalling pathways and stimulating secretion of interleukin-6 and sphingosine-1-phosphate, possibly due to increased ceramidase activity of adiponectin receptors. AdipoRon, an adiponectin analogue, did not show comparable effects. In a mouse model of peritoneal fibrosis, resolvin D1 decreased the proportion of polymorphonuclear cells in the systemic circulation, whereas locally in the abdominal wall it had no effect on inflammation and fibrosis. It also downregulated the expression of chemerin, an important regulator of inflammation that binds to the same receptor as resolvin E1. In our work, we show that oxidative stress and increased migratory potential of monocytes in SSc correspond to the clinical picture of the disease. We show that adiponectin has effects on muscle inflammation and regeneration and that resolvin D1 has the potential to promote resolution of inflammation by regulating the expression of other resolvin receptor ligands.

Enhitreje su bliski srodnici gujavica te modelni organizmi u ekotoksikološkim istraživnjima tla. Iako imaju važnu ulogu u ekološkim sustavima tla, djelovanje pojedinih zagađivala, posebice strobilurinskih fungicida nedovoljno je istraženo. Stoga je cilj ovog doktorskog rada nadopuniti postojeća istraživanja novim znanjima o utjecaju strobilurinskih fungicida na enhitreje vrsta E. crypticus i E. albidus. Razvijena je metoda za mjerenje promjena aktivnosti mehanizma multiksenobiotske rezistentnosti čime se dokazala prisutnost ovog mehanizma kod enhitreja i mogućnost njegova korištenja kao biomarkera zagađenja. Nadalje, uočeno je negativno djelovanje strobilurinskih fungicida na preživljavanje, reprodukciju i uspjeh izlijeganja enhitreja. Prikupljena inicijalna znanja o djelovanju aktivne tvari i različitih pripravaka na biokemijske i stanične biomarkere ukazuju na moguće mehanizme djelovanja strobilurinskih fungicida naglašavajući važnost sveobuhvatnog pristupa. Enchytraeids are close relatives of earthworms and model organisms of soil ecotoxicology. Although they play an important role in soil ecosystems, the effects of certain pollutants, especially strobilurin fungicides, have not been sufficiently investigated. Therefore, the main objective of this doctoral thesis is to supplement existing research with new knowledge about the influence of strobilurin fungicides on the enchytraeid species E. crypticus and E. albidus. The development of a method for measuring changes in the activity of the multixenobiotic resistance mechanism proved the presence of this mechanism in enchytraeids and the possibility of its use as a biomarker. Furthermore, a negative effect of strobilurin fungicides was observed on the survival, reproduction, and hatching success of enchytraeids. Initial knowledge collected on the effect of the active ingredient and different formulations on biochemical and cellular biomarkers indicates the possible mechanisms of action of strobilurin fungicides, emphasizing the importance of a comprehensive approach.

Elektromagnetsko se zračenje emitira iz prirodnog okruženja, ali i uporabom industrijskih i svakodnevnih uređaja za bežičnu komunikaciju, stoga su ljudski i životinjski organizmi stalno izloženi zračenju. Tijekom posljednjih godina, zbog brzog tehnološkog napretka, elektromagnetsko zračenje iz umjetnih izvora premašilo je vrijednosti zračenja prirodnog podrijetla. Opća zabrinutost svih nas, zbog sve većeg broja uređaja (mobilnih telefona, prijenosnih računala, Wi-Fi-ja i mikrovalnih pećnica), koji koriste radiofrekvencijsko elektromagnetsko zračenje (RF-EMZ) opravdana je zbog sve brojnijih dokaza o njihovoj štetnosti na žive organizme. Suvremeni uređaji moderne tehnologije emitiraju radiofrekvencijske elektromagnetske valove malih frekvencija koje ljudsko i životinjsko tijelo apsorbira što može imati potencijalne štetne učinke na: mozak, srce, endokrini sustav i reproduktivnu funkciju. Muški je reproduktivni sustav jedno od najosjetljivijih tkiva na RF-EMZ-e. Tako je primjerice, iz trenutno dostupnih studija provedenih in vitro i in vivo, jasno da RF-EMZ-e ima štetne učinke na spermatogenezu, odnosno kakvoću ejakulata ljudi i životinja – broj spermija u ejakulatu, preživljavanje, morfologiju i gibljivost spermija - utječe na stanični metabolizam i endokrini sustav i može prouzročiti genotoksičnost, genomsku nestabilnost i oksidativni stres, a to može prouzročiti neplodnost. Štetni učinci RFEMZ-a dijele se na toplinske i netoplinske. Većinanegativnih bioloških učinaka pripisuje se netoplinskim učincima, a toplinski se učinci nastali RF-EMZ mobilnog telefona, smatraju manje štetnima. Zbog stvaranja prevelike količine reaktivnih kisikovih spojeva u muškom spolnom sustavu hipertermija skrotuma i povećani oksidativni stres mogu biti ključni mehanizmi putem kojih RF-EMZ-e utječe na plodnost muškaraca. Navedeni su i negativni učinci povezani s vremenom korištenja, ponajprije mobilnog telefona. Stoga je cilj ovoga preglednog rada opisati neke od učinaka RF-EMZ-a na muški spolni sustav., Humans and animals are constantly exposed to electromagnetic radiation emitted from the natural environment, and through the use of industrial and everyday devices for wireless communication. In recent years, due to rapid technological progress, electromagnetic radiation from artificial sources has exceeded the values of radiation of natural origin. General concern about the increasing number of devices (mobile phones, laptops, Wi-Fi and microwave ovens) using radiofrequency electromagnetic radiation (RF-EMR) is justified due to increasing evidence of their harm to the living organism. Modern technology devices emit small frequency radiofrequency electromagnetic waves, which are then absorbed by the human and animal bodies and can potentially cause adverse effects on the brain, heart, endocrine system and reproductive function. It is believed that the male reproductive system is one of the most sensitive tissues to RF-EMR. It is clear from the literature that RF-EMR has harmful effects on ejaculate quality indicators (such as spermatozoa count in ejaculate and spermatozoa morphology and motility), affects cellular metabolism and the endocrine system, and causes genotoxicity, genomic instability and oxidative stress, which in turn may result in infertility. The adverse effects of RF-EMR are divided into thermal and non-thermal. Most negative biological effects are attributed to non-thermal effects, while the thermal effects from cell phone radiation are considered to be less harmful. However, scrotum hyperthermia and increased oxidative stress from the formation of excess reactive oxygen compounds in the male reproductive system can be key mechanisms by which RF-EMR affects male fertility. The negative effects associated with the duration of use of mobile phones are known and listed above. Therefore, the aim of this review article is to describe some of the effects of RF-EMR on the male reproductive system.

High-intensity exercise can elicit acute changes in the biochemical and physiological processes in the body of an athlete, including increased oxidative stress and inflammation. The purpose of this study was to explore the effect of acute moderate-intensity exercise on total antioxidant capacity (TAC) and serum levels of anti-inflammatory adiponectin (APN), and inflammatory markers in competitive cyclists. Ten male cyclists (age 15–26 years, body mass index 19.4–24.7 kg/m$^2$) participated in this study. Each subject performed the maximal oxygen uptake test (VO$_{2peak}$) and completed a 10-min cycling exercise at a workload of 50% of the peak of VO$_{2peak}$. Blood samples were collected on three different occasions: after an overnight fasting and at the exercise workloads of 50% and 100% VO$_{2peak}$. We measured APN, TAC, inflammatory markers as well as assessed nutrient and energy intake for each participant. Baseline concentration of serum APN (10.92µg/mL) significantly increased at 50% and at 100% VO$_{2peak}$. In addition, TAC also increased after acute exercise (0.079 vs 0.093 nmol/µL). The concentration of APN at 50% VO$_{2peak}$ positively correlated with the CRP (r = 0.640, p = 0.046) and negatively correlated with TNF-α (r = −0.696, p = 0.025). This test showed that short (10min) and medium-intensity (50% VO$_{2peak}$) exercise activity in trained athletes evoked beneficial antioxidant and anti-inflammatory responses. Importantly, this response correlates with the increase in APN levels thereby showing that highly trained individuals have beneficial responses originating from adipose tissue. Our observations show that a short training at moderate activity can be an important preservative strategy during the recovery training period.

Estrogeni, oksidativni stres in hipoksija so pomembni regulatorji kostne prenove in homeostaze. Poleg pomanjkanja estrogenov, ki so najpogosteje opisan patološki dejavnik, odgovoren za izgubo kostne mase in spremembe v mikroarhitekturi kostnine, ki vodijo v razvoj osteoporozo, postaja vedno bolj jasen tudi prispevek staranja in z njim povezanega oksidativnega stresa. Dobra vaskularizacija kostnega tkiva in s tem ustrezna preskrbljenost s kisikom prav tako igra pomembno vlogo pri normalnem razvoju skeleta in učinkoviti regeneraciji poškodb kostnine. Poleg omenjenih regulatorjev kostne homeostaze igrajo pomembno regulatorno vlogo v kosteh tudi epigenetski mehanizmi. Epigenetika je veda, ki proučuje stabilne in potencialno dedne spremembe v izražanju genov, za katere niso odgovorne spremembe v zaporedju DNA. Med najpomembnejše epigenetske mehanizme uvrščamo posttranslacijske spremembe histonov, posttranskripcijsko uravnavanje z mikroRNA in metilacijo DNA. Najbolj proučevana posttranslacijska sprememba histonov je acetilacija, za katero so odgovorne histonske acetilaze in deacetilaze. Medtem ko acetilacija histonov omogoči in spodbudi izražanje tarčnih genov, deacetilacija histonov transkripcijo zavre. Slednja pogosto predstavlja le prehodno inhibicijo izražanja tarčnih genov, ki pa lahko preide v bolj trajno obliko z metilacijo DNA. To nalogo opravljajo DNA-metiltransferaze, pri čemer pa je demetilacija lahko pasivna ali aktivna. Podobno kot deacetilacija histonov in metilacija DNA igrajo tudi mikroRNA predvsem inhibitorno regulatorno vlogo. Epigenetske spremembe so postale pomemben del raziskav številnih fizioloških in patoloških procesov tudi zato, ker lahko starost, mehanske obremenitve in okoljski dejavniki ravno preko njih vplivajo na izražanje tarčnih genov. Ker je povezava med znanimi regulatorji kostne homeostaze na eni strani in epigenetskimi procesi na drugi strani še vedno slabo raziskana, je bil namen doktorskega dela proučiti, kako estrogeni, oksidativni stres in hipoksija vplivajo na izražanje genov za histonske acetilaze, histonske deacetilaze, DNA-metiltransferaze in mikroRNA ter s tem na epigenetske procese v osteoblastih. V ta namen smo si najprej pripravili primerni eksperimentalni in vitro model, pri čemer smo uporabili osteosarkomsko celično linijo HOS, v katero smo vnesli kodirajoči del gena za estrogenski receptor α. Za proučevanje vpliva estrogenov smo uporabili 17β-estradiol, oksidativni stres smo izzvali z vodikovim peroksidom, hipoksijo pa z dodatkom mimetika hipoksije deferoksamina. Analizo izražanja genov, povezanih z epigenetskimi mehanizmi, smo izvedli dvostopenjsko. Na osnovi poznavanja epigenetskih mehanizmov in kostne biologije smo za presejalno testiranje izbrali 13 genov, ki kodirajo histonske acetilaze, 18 genov, ki kodirajo histonske deacetilaze, 9 genov, ki kodirajo DNA- metiltransferaze, in druge proteine, povezane z metilacijo DNA, 5 genov, značilnih za osteoblastno celično vrsto, in 3 hišne gene ter njihovo izražanje izmerili s pomočjo mrež, ki temeljijo na kvantitativni verižni reakciji s polimerazo v realnem času (qPCR). V potrditveni stopnji smo tiste gene, pri katerih smo zaznali spremembe v izražanju, analizirali še s posameznimi qPCR-testi na vzorcih večjega števila bioloških ponovitev. Rezultate, pridobljene na eksperimentalnem in vitro modelu, smo nato nadgradili in jim dodali poseben pomen z analizo izražanja najbolj obetavnih genov v obsežni zbirki kostnih vzorcev, pridobljenih od bolnikov z osteoporozo ali osteoartrozo, ter kontrol, brez znakov kostnih bolezni. Proučili smo tudi vpliv estrogenov, oksidativnega stresa in hipoksije na izražanje mikroRNA v osteoblastih, pri čemer smo uporabili zelo zmogljivo tehnologijo barvnih kod. V času razvoja in optimizacije našega eksperimentalnega in vitro modela smo se soočili z več izzivi, katerih pomen presega okvire naše študije. Tako smo ugotovili, da tripsinski reagent, pridobljen iz živalskega pankreasa, ki se sicer rutinsko uporablja pri presajanju adherentnih celic, vsebuje RNaze, ki vstopajo v celice z endocitozo in razgradijo njihovo RNA, ki je tako neprimerna za nadaljnje analize. Rešitev smo našli v neposredni lizi celic z reagentom, ki vsebuje fenol in gvanidin, tik pred samo izolacijo RNA ali v uporabi rekombinantnih tripsinskih nadomestkov, ki niso živalskega izvora. Prav tako se je izkazalo, da v osteosarkomski celični liniji HOS estrogenski receptor α ni prisoten niti na ravni mRNA niti na ravni proteinov. Najizraziteje se je izražal z G proteinom sklopljen estrogenski receptor 1, prisotno pa je bilo tudi šibko izražanje estrogenskega receptorja β. V našem eksperimentalnem in vitro modelu, z vnesenim estrogenskim receptorjem α, estrogeni niso vplivali na izražanje izbranih genov, povezanih z epigenetskimi mehanizmi. Izjema je bilo izražanje mikroRNA miR-338-3p. V skladu s tem estrogeni prav tako niso preprečili sprememb, ki sta jih v osteoblastih povzročila oksidativni stres in hipoksija. Oksidativni stres in hipoksija sta značilno vplivala na izražanje HDAC6, HDAC9, SIRT1 in MBD1, hipoksija pa še dodatno na izražanje HAT1, KAT5, HDAC7 in DNMT3A. Oksidativni stres in hipoksija sta prav tako značilno vplivala na izražanje miR-27, miR-34c, miR-133a, miR-148a, miR-155, miR-214-3p, miR-335, miR-503, miR-630 in miR-1246. Kljub temu, da sta imela zelo blag učinek na aktivnost signalne poti WNT/β-katenin, sta jo oba zavrla. Močnejši, vendar nasproten učinek sta imela tudi na sistem RANK/RANKL/OPG. Naši rezultati razkrivajo potencialno povezanost omenjenih signalnih poti z epigenetskimi mehanizmi v osteoblastih. Analiza izražanja genov, povezanih z epigenetskimi mehanizmi, v kostnih vzorcih je še dodatno potrdila izjemen pomen identificiranih genov v fiziologiji in patologiji kostnih celic. HAT1, KAT5, HDAC6, HDAC9, MBD1 in DNMT3A so tako novi kandidatni geni z zelo verjetno vlogo pri osteoporozi in osteoartrozi, na katere bi se morale osredotočiti prihodnje študije. Omenjeni geni prav tako predstavljajo potencialne tarče v osteoblastih, na katerih bi lahko temeljila prihodnja generacija zdravilnih učinkovin z anabolnim delovanjem na skelet. Estrogens, oxidative stress and hypoxia are important regulators of bone remodeling and homeostasis. In addition to estrogen deficiency, the most often described factor responsible for osteoporosis-associated loss of bone mass and changes in bone microarchitecture, the contribution of aging and associated oxidative stress is becoming increasingly accepted. Sufficient vascularization and oxygen supply to bone tissues are also important for proper skeletal development and efficient bone regeneration and repair. In addition to the described regulators of bone homeostasis, epigenetic mechanisms also play an important regulatory role in bone. Epigenetics is the study of stable and potentially heritable changes in gene expression not associated with changes in DNA sequence. The most important epigenetic mechanisms include posttranslational histone modifications, posttranscriptional regulation through microRNA and DNA methylation. Acetylation is the most studied posttranslational histone modification mediated by histone acetylases and deacetylases. While histone acetylation enables and stimulates target gene expression, histone deacetylation inhibits gene transcription. The latter often represents only a transient inhibition of target gene transcription that can become more permanent through DNA methylation, mediated by DNA methyltransferases. Of note, DNA demethylation can be either passive or active. Similarly to histone deacetylation and DNA methylation, microRNAs also play mostly inhibitory regulatory role. Epigenetic changes have become an important part of research in several physiological and pathological processes, since they represent a mean by which aging, mechanical stress and environmental factors influence target gene transcription. The association between known regulators of bone homeostasis and epigenetic processes is still poorly understood, therefore the aim of the thesis was to examine how estrogens, oxidative stress and hypoxia influence the expression of genes encoding histone acetylases, histone deacetylases, DNA methyltransferases and microRNAs and through that epigenetic processes in osteoblasts. To this end a suitable experimental in vitro model was prepared by transfecting an osteosarcoma cell line HOS with the gene encoding estrogen receptor α. 17β-estradiol was used to study the influence of estrogens, hydrogen peroxide to induce oxidative stress and hypoxia mimetic deferoxamine to establish hypoxia. The expression of genes associated with epigenetic mechanisms was analyzed in a stepwise manner. Based on our knowledge of epigenetic mechanisms and bone regulation, 13 genes encoding histone acetylases, 18 genes encoding histone deacetylases, 9 genes encoding proteins associated with DNA methylation, 5 osteoblast lineage-associated genes and 3 housekeeping genes were selected for gene expression screening using qPCR arrays. In the confirmation step, the most affected genes were analyzed in individual qPCR analyses on samples from several biological replicates. The results obtained in our experimental in vitro model were further substantiated with the expression analysis of the most promising candidate genes on our extensive collection of bone tissue samples obtained from patients with osteoporosis and osteoarthritis as well as controls with no signs of skeletal pathologies. The influence of estrogens, oxidative stress and hypoxia on expression of microRNAs in osteoblasts was also analyzed by using a very powerful technology of color codes. During the development and optimization of our experimental in vitro model we were faced with several challenges, the impact of which exceeds the extent of our study. Our work revealed the presence of contaminating ribonucleases in the animal pancreas-derived trypsin reagent, used routinely for subculturing of adherent cell lines. Ribonucleases entered the cells through endocytosis causing degradation of RNA rendering it useless for further analysis. Our solution was cell lysis with a phenol-guanidine-based reagent directly in the cell culture vessel prior to RNA isolation, or the use of an animal-origin free recombinant trypsin replacement reagent. In addition, we observed a complete lack of estrogen receptor α mRNA and protein in the osteosarcoma cell line HOS. Interestingly, G protein-coupled estrogen receptor 1 gene showed the highest level of expression accompanied by a weak expression of estrogen receptor β. In our experimental in vitro model transfected with estrogen receptor α estrogens had no influence on expression of selected genes associated with epigenetic mechanisms. Expression of microRNA miR-338-3p was the only exception. In accordance with these observations estrogens were not able to prevent changes induced by oxidative stress and hypoxia in osteoblasts. Oxidative stress and hypoxia significantly affected expression of HDAC6, HDAC9, SIRT1 and MBD1, while hypoxia additionally affected expression of HAT1, KAT5, HDAC7 and DNMT3A. Oxidative stress and hypoxia also significantly affected the expression of miR-27, miR-34c, miR-133a, miR-148a, miR-155, miR-214-3p, miR-335, miR-503, miR-630 and miR-1246. Despite having only a mild effect on the activity of WNT/β-catenin signaling pathway, both stimuli exhibited an inhibitory effect. They exhibited a more substantial although opposite effect on RANK/RANKL/OPG system. Our results revealed a potential link between the two signaling pathways and epigenetic mechanisms in osteoblasts. Expression analysis of genes associated with epigenetic mechanisms in bone tissue samples further underlined the importance of identified genes in the physiology and pathology of bone cells. HAT1, KAT5, HDAC6, HDAC9, MBD1 and DNMT3A represent new candidate genes with a very likely role in osteoporosis and osteoarthritis and should be the focus of studies in the future. These genes also represent potential drug targets in osteoblasts that could form the basis for the next generation of active substances with an anabolic effect on the skeleton.

Diazinon (DZN) je organofosfatni pesticid koji se široko koristio u poljoprivredi. Svoje toksične efekte ostvaruje inhibicijom acetilholinesteraze (AChE) i indukovanjem oksidativnog stresa. U ovoj studiji, Wistar pacovi su oralno tretirani diazinonom (55 mg/kg) tokom četiri različita perioda: 7, 14, 21 i 28 dana. Posle svakog vremenskog perioda uzorkovano je tkivo jetre za ispitivanje enzimskih i neenzimskih biomarkera oksidativnog stresa. Analizirane su aktivnosti enzima AChE, superoksid-dizmutaze 1 (SOD1), katalaze (CAT), mijeloperoksidaze (MPO) i paraoksonaze 1 (PON1). Stepen oštećenja ćelijske membrane i proteina praćen je određivanjem koncentracije malondialdehida (MDA) i karbonilnih grupa (CO). Najveća inhibicija AChE zabeležena je između 7. (p

Background: Given the fact that the studies that examined oxidative stress in relation to obesity that included late adolescents are scarce and show inconclusive results we aimed to investigate a wide spectrum of nitro-oxidative stress biomarkers i.e., malondialdehyde (MDA), xanthine oxidase (XO), xanthine oxidoreductase (XOD), xanthine dehydrogenase (XDH), advanced oxidation protein products (AOPP) and nitric oxide products (NOx), as well as an antioxidative enzyme, i.e., catalase (CAT) in relation with obesity in the cohort of adolescent girls ages between 16 and 19 years old. Methods: A total of 59 teenage girls were included in this cross-sectional study. Binary logistic regression analysis was performed to examine possible associations between bio- chemical and nitro-oxidative stress markers and body mass index (BMI). Results: There were not significant differences between oxidative stress markers between normal weight and overweight/obese girls (i.e., AOPP, XOD, XO, XDH) and CAT, except for MDA (p

Background: Multiple sclerosis (MS) is characterized by inflammation, demyelination and axonal degeneration. Oxidative stress (OS) plays a significant role in the pathogenesis of the disease. The aim of the study was to examine the association between OS and smoking on the MS development. Methods: The study included 175 patients with relapsing- remitting multiple sclerosis (RRMS) (76 males, 99 females) and 254 healthy subjects (81 males and 173 females). Oxidative stress biomarkers in serum, Total Antioxidant Status (TAS) and Total Oxidative Status (TOS) were determined spectrophotometrically. Oxidative Stress Index (OSI) was calculated as the ratio of TOS and TAS. Urinary 8-oxo- 7,8-dihydro-2’-deoxyguanosine were determined by HPLC-MS/MS and expressed as 8-oxodG/creatinine. Results: In females with RRMS were higher TOS, OSI and 8-oxodG/creatinine than in females in control group. The group of males with RRMS had lower level of TAS than the males in control group. Higher levels of 8-oxodG/creatinine was obtained in active, passive and former smokers with RRMS than in control group with the same exposition to tobacco smoke. Independent predictors of MS are passive smoking, increased OSI and increased levels of urinary 8-oxodG/creatinine. Conclusions: Our results demonstrate that the OS parameters should be included in the assessment of the risk for MS development. Due to the more sensitivity to oxidative stress, females may be at higher risk of MS development. This data indicates the importance of introducing the antioxidant therapy as a complementary treatment in patients with RRMS. Uvod: Multipla skleroza (MS) se karakteriše upalom, demijelinizacijom i degeneracijom aksona. Oksidativni stres (OS) igra značajnu ulogu u patogenezi bolesti. Cilj studije je bio da se ispita povezanost OS i pušenja na razvoj MS. Metode: Studija je obuhvatila 175 pacijenata sa relapsnoremitentnom multiplom sklerozom (RRMS) (76 muškaraca, 99 žena) i 254 zdrava ispitanika (81 muškarac i 173 žene). Biomarkeri oksidativnog stresa u serumu, ukupni antioksidativni status (TAS) i ukupni oksidativni status (TOS) su određeni spektrofotometrijski. Indeks oksidativnog stresa (OSI) je izračunat kao odnos TOS i TAS. Urinarni 8-okso7,8-dihidro-2'-deoksiguanozin je određen HPLC-MS/MS i izražen kao 8-oksoG/kreatinin. Rezultati: Kod žena sa RRMS bili su viši TOS, OSI i 8okodG/kreatinin nego kod žena u kontrolnoj grupi. Grupa muškaraca sa RRMS imala je niži nivo TAS od muškaraca u kontrolnoj grupi. Veći nivoi 8-okodG/kreatinina su dobijeni kod aktivnih, pasivnih i bivših pušača sa RRMS nego u kontrolnoj grupi sa istom izloženošću duvanskom dimu. Nezavisni prediktori MS su pasivno pušenje, povećan OSI i povećani nivoi 8-okodG/kreatinina u urinu. Zaključak: Naši rezultati pokazuju da parametre OS treba uključiti u procenu rizika za razvoj MS. Zbog veće osetljivosti na oksidativni stres, žene mogu biti izložene većem riziku od razvoja MS. Ovi podaci ukazuju na značaj uvođenja antioksidativne terapije kao komplementarnog lečenja kod pacijenata sa RRMS.

Neonatalna hipoksično-ishemična encefalopatija (HIE) je posledica obporodnega pomanjkanja kisika v osrednjem živčevju ter je eden najbolj pogostih vzrokov za trajno nevrološko okvaro otrok. Pri nekaterih otrocih se kasneje razvije cerebralna paraliza (CP). Pri HIE pride do povečanega nastanka reaktivnih kisikovih spojin in aktivacije vnetnih procesov. Genetska variabilnost v teh poteh bi lahko vplivala na nastanek CP po HIE. V raziskavi smo se osredotočili na pogoste polimorfizme v ključnih genih, ki sodelujejo pri antioksidativni obrambi in pri vnetnem odgovoru po HIE. Vključili smo 90 otrok z zmerno in hudo HIE. S kompetitivnim alelno-specifičnim PCR smo določili polimorfizme v izbranih genih za antioksidativne encime (SOD2 rs4880, CAT rs1001179, GPX1 rs1050450) in provnetne dejavnike (NLRP3 rs35829419, CARD8 rs2043211, IL1B rs1143623, IL1B rs16944, IL1B rs1071671, TNF rs1800629). Pri otrocih, ki so bili zdravljeni s TH in so po rojstvu opravili MR slikanje glave, je bila stopnja možganske okvare opredeljena po klasifikaciji Rutheford. Povezanost polimorfizmov z razvojem CP po HIE in z izidom na MR smo analizirali z uporabo standardnih statističnih testov. Povezave med izbranimi polimorfizmi in nastankom CP nismo dokazali. V skupini otrok, ki ni bila zdravljena s TH, je bil polimorfen alel NLRP3 rs35829419 nominalno značilno povezan z večjim tveganjem za nastanek CP (p = 0,034). V skupini, zdravljeni s TH, pa preučevani polimorfizmi niso bili povezani s pojavom CP. V skupini, zdravljeni s TH, je bil polimorfni alel CARD8 rs2043211 nominalno značilno povezan z normalno/blago možgansko poškodbo na MR glave (p = 0,040), IL1B rs1143623 C alel pa je bil nominalno povezan z zmerno/hudo možgansko poškodbo na MR (p = 0,040). Hipoteze, da so polimorfizmi v izbranih genih povezani s pojavom CP po HIE, nismo potrdili. Vpliv polimorfizmov na izid po HIE je bil do sedaj slabo poznan, zato rezultati predstavljajo izhodišče za nadaljnja raziskovanja na tem področju z večjim številom preiskovancev. Neonatal hypoxic-ischemic encephalopathy (HIE) results from peripartum or intrapartum brain tissue hypoxia and ischemia and is a common cause of long-term neurologic sequelae, including cerebral palsy (CP). Hypoxic-ischemic (HI) insult results in oxidative stress and activation of inflammatory processes. Genetic variability in these pathways could influence the development of CP after HIE. The aim of our study was to evaluate the association of the selected single nucleotide polymorphisms (SNPs) in the genes involved in response to oxidative stress and inflammation with the development of CP after HIE. Ninety children with moderate and severe HIE were included in the study. Genotyping using competitive allele-specific PCR was performed for polymorphisms in genes encoding antioxidant enzymes (SOD2 rs4880, CAT rs1001179, GPX1 rs1050450) and proinflammatory factors (NLRP3 rs35829419, CARD8 rs2043211, IL1B rs1143623, IL1B rs16944, IL1B rs1071676, TNF rs1800629) that could contribute to the development of CP after HIE. In children treated with TH where MRI was performed, it was classified according to the Rutheford classification. Standard statistical tests were used to evaluate the association of selected SNPs with the development of CP. No association between the selected SNPs and the development of CP was found. Polymorphic NLRP3 rs35829419 allele was nominally significantly associated with the development of CP in children who were not treated with TH (p = 0.034). None of the SNPs were associated with CP in children treated with TH. Among children treated with TH polymorphic allele CARD8 rs2043211 was nominally significantly associated with normal/mild MRI overall findings (p = 0.040) and IL1B rs1143623 C allele was nominally significantly associated with moderate/severe MRI overall findings (p = 0.040). The association of genetic variability with the outcome after HIE has so far not been well established, therefore the results of our study represent foundations for future research in larger studies.

V današnjem času smo zaradi hitrega načina življenja izpostavljeni stresu, ki se ga začnemo zavedati šele ob izbruhu določenih bolezni. Stres, ki ga povzročajo reaktivne kisikove in dušikove zvrsti, imenujemo oksidativni stres. Reaktivne zvrsti so zelo nevarne in škodljive, kadar nastajajo prekomerno v organizmu, saj lahko poškodujejo deoksiribonukleinsko kislino, lipide in proteine. Nevarnost poškodb bioloških molekul lahko preprečimo z vnosom antioksidantov, ki zamejijo oksidativne poškodbe biomolekul. Številni antioksidanti so prisotni v sadju in zelenjavi, zato z uživanjem le-teh lahko pripomoremo k boljši antioksidativni obrambi. V magistrski nalogi smo ovrednotili antioksidativno kapaciteto izbranim kinazolinonom z metodo antioksidativne kapacitete redukcije bakrovih(II) ionov (CUPRAC). Metoda temelji na redukciji bakrovega(II) iona v bakrov(I) ion s spojino, ki jo vrednotimo. Pri redukciji se modra barva kompleksa bakrovega(II) iona z neokuproinom spremeni v oranžno-rumeno barvo. Kot standard smo uporabili vodotopni analog α-tokoferola Trolox®. Ugotovili smo, da nekatere testirane spojine izkazujejo boljšo antioksidativno kapaciteto od Troloxa (5 spojin), spet druge pa slabšo (spojine 8, 9, 10) oziroma sploh ne izkazujejo antioksidativnih lastnosti (spojina 1). Za vrednotenje rezultatov smo iz molarnega absorpcijskega koeficienta (ε) spojine in molarnega absorpcijskega koeficienta (ε) standarda (Trolox) izračunali razmerje antioksidativne kapaciteta ekvivalentne Troloxu TEACCUPRAC. Ugotovili smo, da na antioksidativno kapaciteto kinazolinonov vpliva položaj in število fenolnih -OH skupin. Več kot je hidroksilnih skupin vezanih na aromatski obroč, večja je antioksidativna kapaciteta, boljši je antioksidant. Prav tako smo ugotovili, da prisotnost metoksi skupine v odvisnosti od položaja glede na hidroksilno skupino bistveno poveča antioksidativno kapaciteto. Zamenjava metoksi skupine z etoksi skupino pa ni bistveno vplivala na povečanje antioksidativne kapacitete. Nowadays, due to our fast-paced lifestyle, we are exposed to stress, which we only become aware of when certain diseases break out. The stress caused by reactive oxygen and nitrogen species is called oxidative stress. Reactive species are very dangerous and harmful when they are produced excessively in the body, as they can damage deoxyribonucleic acid, lipids and proteins. The risk of damage to biological molecules can be prevented by taking antioxidants that limit oxidative damage to biomolecules. Many antioxidants are present in fruits and vegetables, so by eating them we can contribute to a better antioxidant defence. In the Master's thesis, we evaluated the antioxidant capacity of selected quinazolinones using the copper(II) ion reduction antioxidant capacity (CUPRAC) method. The method is based on the reduction of copper(II) ion to copper(I) ion with the compound being evaluated. During reduction, the blue colour of the copper(II) ion complex with neocuproine changes to an orange-yellow colour. A water-soluble analog of α-tocopherol Trolox® was used as a standard. We found that some of the tested compounds show better antioxidant capacity than Trolox (5 compounds), while others show worse (compounds 8, 9, 10) or no antioxidant properties at all (compound 1). To evaluate the results, we calculated the ratio of antioxidant capacity equivalent to Trolox TEACCUPRAC from the molar absorption coefficient (ε) of the compound and the molar absorption coefficient (ε) of the standard (Trolox). We found that the antioxidant capacity of quinazolinones is influenced by the position and number of phenolic -OH groups. The more hydroxyl groups attached to the aromatic ring, the greater the antioxidant capacity, the better the antioxidant. We also found that the presence of the methoxy group significantly increases the antioxidant capacity, depending on the position relative to the hydroxyl group. Replacing the methoxy group with an ethoxy group did not significantly affect the increase in antioxidant capacity.

Misaligned teeth have a tremendous impact on oral and dental health, and the most efficient method of correcting the problem is orthodontic treatment with orthodontic appliances. The study was conducted to investigate the metal composition of selected orthodontic alloys, the release of metal ions, and the oxidative consequences that the metal ions may cause in the cell. Different sets of archwires, stainless steel brackets, and molar bands were incubated in artificial saliva for 90 days. The composition of each orthodontic material and quantification of the concentration of metal ions released were evaluated. Metal ion mixtures were prepared to determine the occurrence of oxidative stress, antioxidant enzyme defense system, and oxidative damage to proteins. The beta titanium alloy released the fewest metal ions and did not cause oxidative stress or protein damage. The metal ions from stainless steel and the cobalt-chromium alloy can cause oxidative stress and protein damage only at high concentrations. All metal ions from orthodontic alloys alter the activity of antioxidant enzymes in some way. The determined amounts of metal ions released from orthodontic appliances in a simulated oral environment are still below the maximum tolerated dose, and the concentrations of released metal ions are not capable of inducing oxidative stress, although some changes in antioxidant enzyme activity were observed at these concentrations.

Misaligned teeth are often corrected with fixed orthodontic appliances. Prolonged stay of the orthodontic appliance in the oral cavity leads to corrosion and wear of the material, from which metal ions and nanoparticles can be released that catalyze reactive oxygen species-generating reactions, which in turn lead to oxidative stress. The objective of this work was to investigate the level of selected systemic oxidative stress parameters during orthodontic treatment, the composition of selected orthodontic alloys, the release of metal ions, and the oxidative consequences that the metal ions may have on the model organism Saccharomyces cerevisiae (S. cerevisiae). The work also aimed to investigate the effects of nanoparticles on the human gingival cell line (HGF). An increase in systemic oxidative stress levels was detected in the capillary blood of orthodontic patients only after 24 hours, after which oxidative stress parameters normalized. The release of metal ions from orthodontic appliances into artificial saliva is constant, but the metal concentrations released are still below the maximum tolerated daily dose. Only high metal ion concentrations were able to generate large amounts of reactive oxygen species in the yeast S. cerevisiae, which the antioxidant system was unable to regulate adequately, resulting in oxidative stress and its damage to biological molecules. The toxicity of nanoparticles and the ability to generate reactive oxygen species in HGF mainly depend on the type, concentration and properties of nanoparticles. We have shown that metal ions from orthodontic appliances are released at such low concentrations that they cannot induce oxidative stress in the yeast S. cerevisiae, although some change in the antioxidant activity of the enzyme was observed. Of the selected nanoparticles, the WS2 nanoparticle was the least toxic to HGF. Malokluzija se pogosto odpravi s nesnemnimi ortodontskimi aparati. Dolgotrajna izpostavljenost ortodontskega aparata v ustnem okolju vodi do korozije in obrabe materiala, iz katerega se lahko sprostijo kovinski ioni in nanodelci, ki katalizirajo reakcije nastanka reaktivnih kisikovih zvrst in posledično povzročijo oksidativni stres. Namen disertacije je bil raziskati sistemske spremembe parametrov oksidativnega stresa med ortodontskim zdravljenjem, sestavo izbranih ortodontskih zlitin, sproščanje kovinskih ionov in oksidativne posledice, ki jih lahko imajo kovinski ioni na modelni organizem Saccharomyces cerevisiae (S. cerevisiae). Namen disertacije je bil tudi raziskati učinke nanodelcev na celično linijo fibroblastov človeške dlesni (HGF). Povečanje ravni sistemskega oksidativnega stresa je bilo mogoče opaziti v kapilarni krvi ortodontskih pacientov šele po 24 urah, nato pa se parametri oksidativnega stresa normalizirajo. Sproščanje kovinskih ionov iz ortodontskih aparatov v umetno slino je konstantno, vendar so koncentracije sproščenih kovin še vedno pod maksimalno dovoljeno dnevno dozo. Le visoke koncentracije kovinskih ionov so sposobne ustvariti velike količine reaktivnih kisikovih zvrst v kvasovki S. cerevisiae, katerih antioksidativni sistem ni zmožen uravnati, in zato prihaja do oksidativnega stresa in oksidativnih poškodb molekul. Toksičnost nanodelcev in sposobnost ustvarjanja reaktivnih kisikovih zvrsti v celični liniji HGF je odvisna od vrste, koncentracije in lastnosti nanodelcev. Dokazali smo, da se iz nesnemnih zobnih aparatov sproščajo kovinski ioni, čigar koncentracije so prenizke, da bi lahko povečale nivo ROS in inducirale oksidativni stres v kvasovki S. cerevisiae, čeprav so bile opažene nekatere spremembe v aktivnosti antioksidativnih encimov. Izmed izbranih nanodelcev so se nanodelci WS2 izkazali kot najmanj toksični v celični liniji HGF.

Mediteranski šumski ekosustavi u Hrvatskoj su od velike vaţnosti zbog ekoloških funkcija koje pruţaju. Zbog velike osjetljivosti ove regije na oneĉišćujuće tvari prisutnih u atmosferi, bitno je pratiti stanje šumskih ekosustava te kako oni reagiraju na njih. Cilj je bio uspostaviti ekološko praćenje u mediteranskim šumama, odrediti koncentracije iona prispjelih atmosferskim taloţenjem, koncentracije prizemnog ozona i fiziološke parametre stabla, odrediti biokemijske pokazatelje oksidativnog stresa te utvrditi uzroĉno-posljediĉne veze izmeĊu fizioloških parametara stabala i atmosferskih unosa. Sva mjerenja, uzorkovanja i analize raĊena su prema priruĉnicima MeĊunarodnog programa za procjenu i motrenje utjecaja zraĉnog oneĉišćenja na šume (UNECE - ICP Forests). Najveće koliĉine atmosferskoga taloţenja utvrĊena su u šumi hrasta crnike. Opterećenja dušikovim spojevima nisu prošla literaturne granice. Koncentracija ozona na svim plohama bila su blizu ili dosegla 100 ppb. Najmanja osutost utvrĊena je na plohi hrasta crnike s < 10 % dok je na ostale tri bila > 40 %. Najveći postotak prirasta bio na plohi alepskog bora. Ispitivanjem odnosa okolišnih varijabli i pokazatelja zdravstvenog stanja šuma (osutost i prirast) utvrdilo se da je osutost znaĉajno povezana sa sadrţajem vode u tlu, dok je prirast bio povezan s sunĉevim zraĉenjem, vlagom i sa parametrima ozona. Korištenjem metode analize sluĉajne šume (eng. Random forest analysis) naglasile su da su parametri ozona bili najvaţniji prediktori koji utjeĉu na rast stabala. UtvrĊene su znaĉajno manje koliĉine klorofila u hrastovim vrstama u znaĉajno osutim stablima (osutost > 25 %), dok je za borove vrste utvrĊeno bilo obrnuto. Akumulirani H2O2 odrazio se na prisutnost lipidne peroksidacije u svim vrstama. Znaĉajne razlike utvrĊene su za znaĉajno osuta stabla hrasta crnike i alepskog bora dok je za hrast medunac utvrĊeno za stabla male osutosti. UtvrĊene su aktivacije antioksidativnih enzima u svim vrstama. Aktivacija enzima askorbat peroksidaze mogla bi se smatrati kao indikatorom stresa za ispitivane vrste. Iako nisu uoĉena vidljiva oštećenja, utvrĊeno je da su biljke bile pod stresom. Mediterranean forest ecosystems in Croatia are of very high significance because of ecological functions they provide. Due to the high sensitivity of this region to air pollutants present in the atmosphere, it is important to monitor the state of forest ecosystems and how they react to them. The aims of this research were to establish environmental monitoring in Mediterranean forest ecosystems, to determine the concentrations of atmospheric deposition and ground-level ozone, the physiological parameters of trees, to determine biochemical indicators of oxidative stress caused by abiotic sources of stress and finally to establish cause-and-effect relationships between physiological parameters of trees and atmospheric inputs. All measurements, sampling and analysis were done according to International co-operative programme on assessment and monitoring of air pollution effects on forests (ICP Forests) manuals. The highest amount of atmospheric deposition was in the holm oak forest. Nitrogen critical loads did not cross literature limit. Ozone on all four plots was found to be close to or reached 100 ppb. The lowest defoliation was determined on the holm oak plot with < 10% while on the other three it was > 40%, and the highest growth percentage was on Aleppo pine plot. The relationship between environmental variables and forest health indicators (defoliation and growth) was found that defoliation was significantly correlated with soil water content at different depths, while growth was correlated with solar radiation, moisture and with all ozone parameters. Using random forest analysis highlighted that ozone parameters were the most important predictors influencing the tree growth. Significantly lower contents of chlorophyll were found in oak species in significantly defoliated trees (defoliation > 25%) and for pine species was vice versa. Accumulated H2O2 was reflected in the presence of lipid peroxidation in all species. Significant differences were found for significantly defoliated holm oak and Aleppo pine trees, while for pubescent oak it was found for low defoliated trees. Activation of all examined antioxidative enzymes in all species was determined. Ascorbate peroxidase enzyme activity could be considered as an indicator of stress in trees for examined species. The plants were found to be under stress although no visible damage was observed.

Nanotehnologija danas ima ulogu rastuće važnosti u razvoju novih industrijskih i medicinskih proizvoda. Nanočestice srebra (nanoAg) jedne su od najviše istraživanih tipova nanomaterijala za potencijalnu primjenu u medicini zbog svojih izraženih antimikrobnih svojstava. Njihova uporaba u medicinskom kontekstu ograničena je zbog štetnog utjecaja kojeg mogu imati na ljudske stanice i tkiva. Iako je toksičnost nanočestica srebra dobro poznata i istražena, manjkaju informacije o kombiniranim biološkim učincima nanoAg i drugih materijala ili nanomaterijala. Za razvoj novih, sigurnih uređaja i pomagala u kojima se koristi nanoAg, nužno je ispitati zdravstvene rizike koji mogu nastati međudjelovanjem nanoAg i materijala koji se primjenjuju u tim proizvodima. Polistiren je biološki inertan materijal široke industrijske i medicinske primjene, te su zbog toga nanočestice polistirena (nanoPS) odabrane za ispitivanje učinka mješavina nanočestica na modelne stanice ljudskog imunosnog sustava, Jurkat i THP-1. Nanočestice su karakterizirane metodama atomske mikroskopije sila te dinamičkog i elektroforetskog raspršenja svjetlosti. Analiziran je utjecaj nanoAg i nanoPS na preživljenje stanica, pokretanje apoptoze, parametre oksidativnog stresa, oštećenje DNA, pokretanje upalnog odgovora te promjene mehaničkih svojstava stanica. Unos nanočestica vizualiziran je konfokalnim mikroskopom i kvantificiran protočnim citometrom. Pokazano je da nanoAg i nanoPS ne stupaju u interakcije u vodenoj suspenziji ili u staničnom mediju, te da su mješavine nanoAg i nanoPS imale štetniji učinak na stanice Jurkat i THP-1 nego pojedinačne vrste nanomaterijala. Ovi rezultati važan su doprinos razumijevanju učinaka nanomaterijala na ljudsko zdravlje i razvoju novih i sigurnih primjena nanotehnologije u medicini. Nanotechnology has a role of growing importance in development of novel industrial and medical applications. Silver nanoparticles (nanoAg) are some of the most researched types of nanomaterials for potential use in medicine due to their significant antimicrobial properties. Their use in medical applications is limited due to their potential toxicity to human cells and tissue. Although the toxicity of silver nanoparticles is well known, there is a distinct lack of information on combined biological effects of nanoAg and other types of materials or nanomaterials. In order to develop safe nanoAg-enabled products, it is paramount to determine if interactions of nanoAg with other materials could pose additional health hazard compared to nanoAg alone. Polystyrene is a biologically inert material with widespread use in industrial and medical applications, and polystyrene nanoparticles (nanoPS) were used to assess the effects of nanometrial mixtures on model immune cell lines, Jurkat and THP-1. Nanoparticles were characterized using atomic force microscopy, as well as dynamic and electrophoretic light scattering techniques. The effects of nanoAg and nanoPS on cell survival, induction of apoptosis, oxidative stress parameters, DNA damage, immune response and changes in cell mechanical properties were analyzed. Nanoparticle uptake was visualized with confocal microscope and quantified using flow cytometry. Results have shown that nanoAg and nanoPS do not interact in water suspensions or in cell culture media, and that nanoAg/nanoPS mixtures had an augmented, detrimental impact on Jurkat and THP-1 cells compared to individual nanoparticle types. These results present an important contribution for understanding impact of nanomaterials on human health, and for development of novel and safe applications of nanotechnology in medicine.

Uvod: Problem slepote kot posledice glavkoma se povečuje zaradi staranja prebivalstva. Nastanek in napredovanje primarnega glavkoma z odprtim zakotjem (POAG) sta povezana z oksidativnim stresom, vnetjem in apoptozo ganglijskih celic mrežnice. Različni dejavniki lahko povzročijo večje nastajanje reaktivnih kisikovih spojin in sproščanje mediatorjev imunsko-vnetnega odziva. Matriksne metaloproteinaze (MMP) so encimi, ki sodelujejo pri preoblikovanju izvenceličnega matriksa v trabekulumu in ciliarniku ter na ta način uravnavajo očesni tlak (IOP). Edini dokazano učinkovit način zdravljenja glavkoma je znižanje IOP. Z zdravili pogosto ne dosežemo zadostnega znižanja IOP, pri nekaterih bolnikih pa kljub nizkemu IOP bolezen napreduje. Latanoprost je prvi prostaglandinski analog, ki ga evropske glavkomske smernice, skupaj s selektivno lasersko trabekuloplastiko, priporočajo kot prvi izbor za zdravljenja glavkoma. Zaenkrat ne poznamo kliničnih ali bioloških označevalcev, s katerimi bi lahko napovedali tveganje za nastanek POAG, njegov potek ter odgovor na določeno vrsto zdravljenja. Hipoteze: Genetska variabilnost lahko vpliva na potek POAG. Farmakogenetska variabilnost napoveduje odgovor na zdravljenje z latanoprostom in s selektivno lasersko trabekuloplastiko (SLT) pri bolnikih s POAG in očesno hipertenzijo (OHT). Z ugotavljanjem genetskih označevalcev lahko napovemo, katero zdravljenje bo pri posameznem bolniku učinkovitejše in povezano z manj neželenimi učinki. Metode: V raziskavo smo vključili 307 bolnikov s POAG ali z OHT, starejših od 40 let in 339 kontrol. V retrospektivnem delu smo pri bolnikih in kontrolah analizirali polimorfizme posameznih nukleotidov (SNP) v genih, ki so vključeni v obrambo pred oksidativnim stresom (GSTM1, GSTT1, GSTP1, SOD2, CAT in GPX1), vnetne poti (TNF, IL1B in IL6) ter delovanje encimov MMP (MMP2, MMP3, MMP9, MMP14). V prospektivni del raziskave smo vključili nezdravljene bolnike, ki smo jih naključno razvrstili za zdravljenje z latanoprostom ali s SLT ter ovrednotili odgovor na zdravljenje po 1,5 meseca. Pri vseh bolnikih smo analizirali SNP v genih, ki so vključeni v antioksidativne in vnetne poti ter za encime MMP. Pri bolnikih zdravljenih z latanoprostom smo analizirali tudi SNP v genih, ki sodelujejo pri farmakodinamiki in farmakokinetiki latanoprosta (CES1,CES1P1, BCHE , SLCO2A1, PTGFR). Introduction: The problem of blindness because of glaucoma is increasing due to the aging population. The onset and progression of primary open-angle glaucoma (POAG) is associated with oxidative stress, inflammation, and apoptosis of retinal ganglion cells. Various factors cause increased formation of reactive oxygen species and release of mediators involved in the immune-inflammatory response. Matrix metalloproteinases (MMP) are enzymes involved in the formation of the extracellular matrix in the trabecular meshwork and ciliary body and thus regulate intraocular pressure (IOP). The only proven effective treatment is reduction of IOP. Medication often does not achieve sufficient reduction of IOP, and in some patients the disease progresses despite low IOP. The first prostaglandin analogue latanoprost and selective laser trabeculoplasty (SLT), have been both recommended as the first-choice treatment for POAG by the European Glaucoma Society Guidelines. Currently, there are no clinical or biological markers that would help to predict the risk of POAG, its course or the response to a particular type of treatment. Hypotheses: Genetic variability may influence the course of POAG. Pharmacogenetic variability predicts response to latanoprost and selective laser trabeculoplasty in patients with POAG and ocular hypertension (OHT). By identifying genetic markers, we can predict which treatment will be more effective and associated with fewer side effects in an individual patient. Methods: A total of 307 patients with POAG or OHT older than 40 years and 339 controls were included in the study. In the retrospective part, we analyzed common single nucleotide polymorphisms (SNPs) in the genes involved in the pathways of oxidative stress (GSTM1, GSTT1, GSTP1, SOD2, CAT and GPX1), inflammation (TNF, IL1B and IL6) and in the activity of MMP (MMP2, MMP9, MMP14). The prospective part of the study included treatment-naive patients randomized to treatment with latanoprost or SLT, and evaluated the treatment response after 1.5 months. In these patients we also analyzed SNPs in the genes involved in the oxidative and inflammatory pathways, and in MMP genes. In addition, in the latanoprost treatment group, we analyzed SNPs involved in the pharmacodynamics and pharmacokinetics of latanoprost (CES1, CES1P1, BCHE , SLCO2A1, PTGFR). Results: Homozygotes for GSTM1 gene deletion had a lower risk of developing OHT, while homozygotes for MMP3 rs3025058 had a higher risk of developing OHT. There were more subjects with a higher degree of glaucoma impairment among carriers of at least one GPX1 polymorphic allele, while among carriers of at least one MMP3 polymorphic allele there were more subjects with a lower degree of impairment. The SOD2 rs4880, CAT rs1001179, and TNF 1800629 polymorphisms were associated with an absolute decrease in IOP, whereas carriers of at least one copy of GSTT1 had a lower relative decrease in IOP after latanoprost treatment. IL6 polymorphism was associated with a smaller absolute and relative decrease in IOP after SLT treatment. The clinical-pharmacogenetic model better predicted the effectiveness of IOP reduction (30% and more) in SLT treatment compared to the clinical model, whereas in latanoprost treatment both models had similar predictive value. Conclusion: SNPs in genes involved in oxidative and inflammatory pathways may be potential biological markers for predicting the risk and progression of POAG, as well as the response to latanoprost and SLT treatment. A clinical-pharmacogenetic prediction model could be helpful in selecting a more effective treatment.

Disbalance balance between oxidants and antioxidants is called oxidative stress and could be presented as oxidative stress index (OSI). OSI is determined by the reactive oxygen metabolites (d-ROM test) to assess oxidants and the plasma antioxidant capacity test (PAT test) to measure antioxidants. The aim of the study was to evaluate the predictive value of OSI in the disease COVID-19. d-ROMs results were the highest in the SARS-CoV-2 POSITIVE group (365+/-112), lower in the SARS-CoV-2 NEGATIVE group (314+/-72.4), and the lowest in an INTENSIVE CARE UNIT group (ICU) (277+/-142) U.Carr. PAT test values were the lowest in the SARS-CoV-2 POSITIVE group (2762+/-387), higher in the ICU group (2772 +/-786), and the highest in the SARS-CoV-2 NEGATIVE group (2808+/-470), and are not statistically significantly different (P>0.05), while OSI was: healthy with average value of 49 and the critical ill with average value of 109 (P = 0.016). Cut-offs for predicting ICUs admission was at OSI 62, with 80.0% sensitivity and 68.2% specificity. Neravnovesje med oksidanti in antioksidanti imenujemo oksidativni stres in ga lahko prikažemo kot indeks oksidativnega stresa (OSI). OSI določimo s testom reaktivnih presnovkov kisika (d-ROM) za oceno oksidantov in testom plazemske antioksidativne kapacitete (PAT) za merjenje antioksidantov. Namen študije je bil oceniti napovedno vrednost OSI pri bolezni COVID-19. Rezultati tasta d-ROM so bili najvišji v skupini SARS-CoV-2 pozitivni (365+/-112), nižji v skupini SARS-CoV-2 negativni (314+/-72,4) in najnižji v skupini kritično bolnih v enoti za intenzivno nego (ICU) (277+/-142) U.Carr. Vrednosti testa PAT so bile najnižje v skupini SARS-CoV-2 pozitivni (2762+/-387), višje v skupini kritično bolnih (2772 +/-786) in najvišje v skupini SARS-CoV-2 negativni (2808 +/-470). Skupine se med sabo statistično značilno ne razlikujejo (P>0,05). OSI se statistično značilno razlikuje med zdravimi s povprečno vrednostjo 49 in kritično bolnimi s povprečno vrednostjo 109 (P = 0,016). Določili smo mejno vrednost za napovedovanje sprejema pacienta v enoto intenzivne nege na osnovi analize OSI, in sicer 62, z 80,0 % občutljivostjo in 68,2 % specifičnostjo.

Литература даје ограничене податке о употреби матичњака код пацијената са инфекцијом вирусом хумане дефицијенције (енг. human immunodeficiency virus ‒ HIV), али подаци клиничких студија указују на бенефите примене ове биљке код поремећаја метаболизма угљених хидрата и липида. Циљ рада је био испитати деловање матичњака уз антиретровирусну терапију која садржи ефавиренз, дарунавир или ралтегравир, као и утврдити позитивне ефекте примене матичњака у регулацији метаболичког синдрома. Извршена је прелиминарна и детаљна хемијска анализа дијететског суплемента на бази екстракта листа матичњака. Испитано је антихипергликемично деловање екстракта у in vitro тестовима инхибиције ензима α-амилазе и α-глукозидазе. Поред тога, суплемент на бази екстракта матичњака и чиста розмаринска киселина су у in vitro условима подвргнути ферментацији помоћу пробиотских микроорганизама. Испитивање на пацијентима је укључило пацијенте Клинике за инфективне болести Клиничког центра Војводине који су инфицирани HIV-ом, са присутним метаболичким синдромом. Пацијенти су на основу лека који примају у терапији подељени на 3 групе, а поред прописане антиретровирусне терапије примали су и матичњак у дози од 600 m током 12 недеља. Пацијентима су на почетку и на крају периода испитивања мерени следећи параметри: концентрација розмаринске киселине у крви, концентрација антиретровирусног лека (ефавиренз, дарунавир или ралтегравир) у крви, број копија вируса HIV-а у крви, хематолошки, имунолошки и биохемијски параметри, показатељи оксидативног стреса, активност ензима антиоксидативне заштите, као и антропометријски параметри. Испитани суплемент на бази екстракта окарактерисан је као добар извор фенолних једињења, при чему је најзаступљенија компонента била розмаринска киселина. Утврђена је in vitro инхибиција оба ензима у релативно ниским концентрацијама, уз бољу активност у инхибицији α-глукозидазе. Путем in vitro ферментације показано је да суплемент на бази екстракта подлеже интензивном метаболизму од стране пробиотских сојева; розмаринска киселина у екстракту је стабилнија у поређењу са изолованом формом. Показано је и да ово једињење представља маркер употребе матичњака у плазми, при чему се розмаринска киселина у крви након 12 недеља употребе доминантно налази у облику глукуронида и сулфата. Нису пронађене статистички значајне разлике између концентрација испитиваних лекова одређених пре и после 12 недеља примене матичњака. Није забележено повећање броја копија вируса у крви. Хематолошки и имунолошки параметри који су праћени нису показали статистички значајну промену вредности. У оквиру биохемијских параметара није дошло до статистички значајних промена које се могу повезати са употребом матичњака, са изузетком испитиване групе ефавиренза, где је дошло до статистички значајног повећања аланин аминотрансферазе и аспартат аминотрансферазе, али је повећање наведених ензима било у оквиру физиолошких вредности. Показано је да након 12 недеља примене матичњака долази до статистички значајне редукције параметара оксидативног стреса у испитиваним групама пацијената који су примали ефавиренз и дарунавир, a у испитиваној групи са ефавирензом потврђено је и статистички значајно повећање активности глутатион-S-трансферазе. У оквиру антропометријски параметара уочено је повећање телесне масе након 12 недеља примене матичњака у испитиваној групи са ефавирензом. Овде представљено истраживање показало је да истовремена примена матичњака уз антиретровирусну терапију која укључује један од испитиваних лекова не утиче на концентрације ових лекова. Праћени параметри који су испитивани код пацијената показали су да је примена матичњака у дози од 600 mg током 12 недеља безбедна за пацијенте. Показани позитивни ефекти указују на повољно дејство код пацијената инфицираних HIV-ом са метаболичким синдромом. Literatura daje ograničene podatke o upotrebi matičnjaka kod pacijenata sa infekcijom virusom humane deficijencije (eng. human immunodeficiency virus ‒ HIV), ali podaci kliničkih studija ukazuju na benefite primene ove biljke kod poremećaja metabolizma ugljenih hidrata i lipida. Cilj rada je bio ispitati delovanje matičnjaka uz antiretrovirusnu terapiju koja sadrži efavirenz, darunavir ili raltegravir, kao i utvrditi pozitivne efekte primene matičnjaka u regulaciji metaboličkog sindroma. Izvršena je preliminarna i detaljna hemijska analiza dijetetskog suplementa na bazi ekstrakta lista matičnjaka. Ispitano je antihiperglikemično delovanje ekstrakta u in vitro testovima inhibicije enzima α-amilaze i α-glukozidaze. Pored toga, suplement na bazi ekstrakta matičnjaka i čista rozmarinska kiselina su u in vitro uslovima podvrgnuti fermentaciji pomoću probiotskih mikroorganizama. Ispitivanje na pacijentima je uključilo pacijente Klinike za infektivne bolesti Kliničkog centra Vojvodine koji su inficirani HIV-om, sa prisutnim metaboličkim sindromom. Pacijenti su na osnovu leka koji primaju u terapiji podeljeni na 3 grupe, a pored propisane antiretrovirusne terapije primali su i matičnjak u dozi od 600 m tokom 12 nedelja. Pacijentima su na početku i na kraju perioda ispitivanja mereni sledeći parametri: koncentracija rozmarinske kiseline u krvi, koncentracija antiretrovirusnog leka (efavirenz, darunavir ili raltegravir) u krvi, broj kopija virusa HIV-a u krvi, hematološki, imunološki i biohemijski parametri, pokazatelji oksidativnog stresa, aktivnost enzima antioksidativne zaštite, kao i antropometrijski parametri. Ispitani suplement na bazi ekstrakta okarakterisan je kao dobar izvor fenolnih jedinjenja, pri čemu je najzastupljenija komponenta bila rozmarinska kiselina. Utvrđena je in vitro inhibicija oba enzima u relativno niskim koncentracijama, uz bolju aktivnost u inhibiciji α-glukozidaze. Putem in vitro fermentacije pokazano je da suplement na bazi ekstrakta podleže intenzivnom metabolizmu od strane probiotskih sojeva; rozmarinska kiselina u ekstraktu je stabilnija u poređenju sa izolovanom formom. Pokazano je i da ovo jedinjenje predstavlja marker upotrebe matičnjaka u plazmi, pri čemu se rozmarinska kiselina u krvi nakon 12 nedelja upotrebe dominantno nalazi u obliku glukuronida i sulfata. Nisu pronađene statistički značajne razlike između koncentracija ispitivanih lekova određenih pre i posle 12 nedelja primene matičnjaka. Nije zabeleženo povećanje broja kopija virusa u krvi. Hematološki i imunološki parametri koji su praćeni nisu pokazali statistički značajnu promenu vrednosti. U okviru biohemijskih parametara nije došlo do statistički značajnih promena koje se mogu povezati sa upotrebom matičnjaka, sa izuzetkom ispitivane grupe efavirenza, gde je došlo do statistički značajnog povećanja alanin aminotransferaze i aspartat aminotransferaze, ali je povećanje navedenih enzima bilo u okviru fizioloških vrednosti. Pokazano je da nakon 12 nedelja primene matičnjaka dolazi do statistički značajne redukcije parametara oksidativnog stresa u ispitivanim grupama pacijenata koji su primali efavirenz i darunavir, a u ispitivanoj grupi sa efavirenzom potvrđeno je i statistički značajno povećanje aktivnosti glutation-S-transferaze. U okviru antropometrijski parametara uočeno je povećanje telesne mase nakon 12 nedelja primene matičnjaka u ispitivanoj grupi sa efavirenzom. Ovde predstavljeno istraživanje pokazalo je da istovremena primena matičnjaka uz antiretrovirusnu terapiju koja uključuje jedan od ispitivanih lekova ne utiče na koncentracije ovih lekova. Praćeni parametri koji su ispitivani kod pacijenata pokazali su da je primena matičnjaka u dozi od 600 mg tokom 12 nedelja bezbedna za pacijente. Pokazani pozitivni efekti ukazuju na povoljno dejstvo kod pacijenata inficiranih HIV-om sa metaboličkim sindromom. Literature contains limited data on the use of lemon balm in patients infected with the human immunodeficiency virus (HIV). However, data from clinical studies indicate the benefits of this plant’s use in carbohydrate and lipid metabolism disorders. The aim of this study was to examine the effect of lemon balm when used alongside antiretroviral therapy containing efavirenz, darunavir or raltegravir, as well as to determine the positive effects of the use of lemon balm in metabolic syndrome regulation. Both a preliminary and a detailed chemical analysis of lemon balm leaf extract were performed. The antihyperglycemic activity of the extract was investigated in in vitro α-amylase and α-glucosidase enzyme inhibition assays. Fermentation of the lemon balm extract, as well as of pure rosmarinic acid with probiotic microorganisms, was also performed in vitro. The investigation performed on patients included patients of the Clinic for Infectious Diseases of the Clinical Center of Vojvodina infected with HIV and with a co-occurring metabolic syndrome. The patients were divided into 3 groups based on the medication they received in antiretroviral therapy, and in addition to the prescribed therapy, they received lemon balm in a dose of 600 mg for 12 weeks. At the beginning and at the end of the study period, the following parameters were measured in patients: the concentration of rosmarinic acid in the patients’ blood, antiretroviral drug concentration (efavirenz, darunavir or raltegravir) in the patients’ blood, the number of copies of HIV in the patients’ blood, hematological, immunological and biochemical parameters, oxidative stress, the activity of enzymes of antioxidant protection, and anthropometric parameters. The tested extract was characterized as a good source of phenolic compounds, with rosemary acid being the most abundant compound. The extract showed in vitro inhibition of both enzymes at relatively low concentrations, with better activity in α-glucosidase inhibition. In in vitro fermentation, the extract has been shown to undergo intensive metabolism by probiotic strains; rosmarinic acid in the extract was more stable compared to the pure chemical compound. Blood analysis has shown that rosmarinic acid is a marker of the use of lemon balm in plasma, however, this compound is found in the form of a sulfoglucuronide in the blood after 12 weeks of use. No statistically significant differences were found between the concentrations of the tested drugs before and after 12 weeks of investigation. There was no increase in the number of copies of the virus in the blood. The monitored hematological and immunological parameters did not show a statistically significant change in values. Within the monitored biochemical parameters, there were no statistically significant changes that can be associated with the use of lemon balm, with the exception of the studied group of efavirenz, where there was a statistically significant increase in alanine aminotransferases and aspartate aminotransferases, but within reference values. It was shown that after 12 weeks of application of lemon balm, there was a statistically significant reduction in oxidative stress parameters in the studied groups of efavirenz and darunavir; in addition, a statistically significant increase in glutathione-S-transferase activity was shown in the efavirenz study group. Regarding the parameters of anthropometry, an increase in body weight was shown in the examined group of efavirenz after 12 weeks of application of lemon balm. The research presented here shows that the simultaneous use of lemon balm with antiretroviral therapy that includes one of the tested drugs does not affect the concentrations of these drugs. The monitored parameters in the patients showed that the use of lemon balm for 12 weeks in a dose of 600 mg was safe for patients. The presented positive results indicate a beneficial effect on HIV-infected patients with metabolic syndrome.

Литература даје ограничене податке о употреби матичњака код пацијената са инфекцијом вирусом хумане дефицијенције (енг. human immunodeficiency virus ‒ HIV), али подаци клиничких студија указују на бенефите примене ове биљке код поремећаја метаболизма угљених хидрата и липида. Циљ рада је био испитати деловање матичњака уз антиретровирусну терапију која садржи ефавиренз, дарунавир или ралтегравир, као и утврдити позитивне ефекте примене матичњака у регулацији метаболичког синдрома. Извршена је прелиминарна и детаљна хемијска анализа дијететског суплемента на бази екстракта листа матичњака. Испитано је антихипергликемично деловање екстракта у in vitro тестовима инхибиције ензима α-амилазе и α-глукозидазе. Поред тога, суплемент на бази екстракта матичњака и чиста розмаринска киселина су у in vitro условима подвргнути ферментацији помоћу пробиотских микроорганизама. Испитивање на пацијентима је укључило пацијенте Клинике за инфективне болести Клиничког центра Војводине који су инфицирани HIV-ом, са присутним метаболичким синдромом. Пацијенти су на основу лека који примају у терапији подељени на 3 групе, а поред прописане антиретровирусне терапије примали су и матичњак у дози од 600 m током 12 недеља. Пацијентима су на почетку и на крају периода испитивања мерени следећи параметри: концентрација розмаринске киселине у крви, концентрација антиретровирусног лека (ефавиренз, дарунавир или ралтегравир) у крви, број копија вируса HIV-а у крви, хематолошки, имунолошки и биохемијски параметри, показатељи оксидативног стреса, активност ензима антиоксидативне заштите, као и антропометријски параметри. Испитани суплемент на бази екстракта окарактерисан је као добар извор фенолних једињења, при чему је најзаступљенија компонента била розмаринска киселина. Утврђена је in vitro инхибиција оба ензима у релативно ниским концентрацијама, уз бољу активност у инхибицији α-глукозидазе. Путем in vitro ферментације показано је да суплемент на бази екстракта подлеже интензивном метаболизму од стране пробиотских сојева; розмаринска киселина у екстракту је стабилнија у поређењу са изолованом формом. Показано је и да ово једињење представља маркер употребе матичњака у плазми, при чему се розмаринска киселина у крви након 12 недеља употребе доминантно налази у облику глукуронида и сулфата. Нису пронађене статистички значајне разлике између концентрација испитиваних лекова одређених пре и после 12 недеља примене матичњака. Није забележено повећање броја копија вируса у крви. Хематолошки и имунолошки параметри који су праћени нису показали статистички значајну промену вредности. У оквиру биохемијских параметара није дошло до статистички значајних промена које се могу повезати са употребом матичњака, са изузетком испитиване групе ефавиренза, где је дошло до статистички значајног повећања аланин аминотрансферазе и аспартат аминотрансферазе, али је повећање наведених ензима било у оквиру физиолошких вредности. Показано је да након 12 недеља примене матичњака долази до статистички значајне редукције параметара оксидативног стреса у испитиваним групама пацијената који су примали ефавиренз и дарунавир, a у испитиваној групи са ефавирензом потврђено је и статистички значајно повећање активности глутатион-S-трансферазе. У оквиру антропометријски параметара уочено је повећање телесне масе након 12 недеља примене матичњака у испитиваној групи са ефавирензом. Овде представљено истраживање показало је да истовремена примена матичњака уз антиретровирусну терапију која укључује један од испитиваних лекова не утиче на концентрације ових лекова. Праћени параметри који су испитивани код пацијената показали су да је примена матичњака у дози од 600 mg током 12 недеља безбедна за пацијенте. Показани позитивни ефекти указују на повољно дејство код пацијената инфицираних HIV-ом са метаболичким синдромом., Literatura daje ograničene podatke o upotrebi matičnjaka kod pacijenata sa infekcijom virusom humane deficijencije (eng. human immunodeficiency virus ‒ HIV), ali podaci kliničkih studija ukazuju na benefite primene ove biljke kod poremećaja metabolizma ugljenih hidrata i lipida. Cilj rada je bio ispitati delovanje matičnjaka uz antiretrovirusnu terapiju koja sadrži efavirenz, darunavir ili raltegravir, kao i utvrditi pozitivne efekte primene matičnjaka u regulaciji metaboličkog sindroma. Izvršena je preliminarna i detaljna hemijska analiza dijetetskog suplementa na bazi ekstrakta lista matičnjaka. Ispitano je antihiperglikemično delovanje ekstrakta u in vitro testovima inhibicije enzima α-amilaze i α-glukozidaze. Pored toga, suplement na bazi ekstrakta matičnjaka i čista rozmarinska kiselina su u in vitro uslovima podvrgnuti fermentaciji pomoću probiotskih mikroorganizama. Ispitivanje na pacijentima je uključilo pacijente Klinike za infektivne bolesti Kliničkog centra Vojvodine koji su inficirani HIV-om, sa prisutnim metaboličkim sindromom. Pacijenti su na osnovu leka koji primaju u terapiji podeljeni na 3 grupe, a pored propisane antiretrovirusne terapije primali su i matičnjak u dozi od 600 m tokom 12 nedelja. Pacijentima su na početku i na kraju perioda ispitivanja mereni sledeći parametri: koncentracija rozmarinske kiseline u krvi, koncentracija antiretrovirusnog leka (efavirenz, darunavir ili raltegravir) u krvi, broj kopija virusa HIV-a u krvi, hematološki, imunološki i biohemijski parametri, pokazatelji oksidativnog stresa, aktivnost enzima antioksidativne zaštite, kao i antropometrijski parametri. Ispitani suplement na bazi ekstrakta okarakterisan je kao dobar izvor fenolnih jedinjenja, pri čemu je najzastupljenija komponenta bila rozmarinska kiselina. Utvrđena je in vitro inhibicija oba enzima u relativno niskim koncentracijama, uz bolju aktivnost u inhibiciji α-glukozidaze. Putem in vitro fermentacije pokazano je da suplement na bazi ekstrakta podleže intenzivnom metabolizmu od strane probiotskih sojeva; rozmarinska kiselina u ekstraktu je stabilnija u poređenju sa izolovanom formom. Pokazano je i da ovo jedinjenje predstavlja marker upotrebe matičnjaka u plazmi, pri čemu se rozmarinska kiselina u krvi nakon 12 nedelja upotrebe dominantno nalazi u obliku glukuronida i sulfata. Nisu pronađene statistički značajne razlike između koncentracija ispitivanih lekova određenih pre i posle 12 nedelja primene matičnjaka. Nije zabeleženo povećanje broja kopija virusa u krvi. Hematološki i imunološki parametri koji su praćeni nisu pokazali statistički značajnu promenu vrednosti. U okviru biohemijskih parametara nije došlo do statistički značajnih promena koje se mogu povezati sa upotrebom matičnjaka, sa izuzetkom ispitivane grupe efavirenza, gde je došlo do statistički značajnog povećanja alanin aminotransferaze i aspartat aminotransferaze, ali je povećanje navedenih enzima bilo u okviru fizioloških vrednosti. Pokazano je da nakon 12 nedelja primene matičnjaka dolazi do statistički značajne redukcije parametara oksidativnog stresa u ispitivanim grupama pacijenata koji su primali efavirenz i darunavir, a u ispitivanoj grupi sa efavirenzom potvrđeno je i statistički značajno povećanje aktivnosti glutation-S-transferaze. U okviru antropometrijski parametara uočeno je povećanje telesne mase nakon 12 nedelja primene matičnjaka u ispitivanoj grupi sa efavirenzom. Ovde predstavljeno istraživanje pokazalo je da istovremena primena matičnjaka uz antiretrovirusnu terapiju koja uključuje jedan od ispitivanih lekova ne utiče na koncentracije ovih lekova. Praćeni parametri koji su ispitivani kod pacijenata pokazali su da je primena matičnjaka u dozi od 600 mg tokom 12 nedelja bezbedna za pacijente. Pokazani pozitivni efekti ukazuju na povoljno dejstvo kod pacijenata inficiranih HIV-om sa metaboličkim sindromom., Literature contains limited data on the use of lemon balm in patients infected with the human immunodeficiency virus (HIV). However, data from clinical studies indicate the benefits of this plant’s use in carbohydrate and lipid metabolism disorders. The aim of this study was to examine the effect of lemon balm when used alongside antiretroviral therapy containing efavirenz, darunavir or raltegravir, as well as to determine the positive effects of the use of lemon balm in metabolic syndrome regulation. Both a preliminary and a detailed chemical analysis of lemon balm leaf extract were performed. The antihyperglycemic activity of the extract was investigated in in vitro α-amylase and α-glucosidase enzyme inhibition assays. Fermentation of the lemon balm extract, as well as of pure rosmarinic acid with probiotic microorganisms, was also performed in vitro. The investigation performed on patients included patients of the Clinic for Infectious Diseases of the Clinical Center of Vojvodina infected with HIV and with a co-occurring metabolic syndrome. The patients were divided into 3 groups based on the medication they received in antiretroviral therapy, and in addition to the prescribed therapy, they received lemon balm in a dose of 600 mg for 12 weeks. At the beginning and at the end of the study period, the following parameters were measured in patients: the concentration of rosmarinic acid in the patients’ blood, antiretroviral drug concentration (efavirenz, darunavir or raltegravir) in the patients’ blood, the number of copies of HIV in the patients’ blood, hematological, immunological and biochemical parameters, oxidative stress, the activity of enzymes of antioxidant protection, and anthropometric parameters. The tested extract was characterized as a good source of phenolic compounds, with rosemary acid being the most abundant compound. The extract showed in vitro inhibition of both enzymes at relatively low concentrations, with better activity in α-glucosidase inhibition. In in vitro fermentation, the extract has been shown to undergo intensive metabolism by probiotic strains; rosmarinic acid in the extract was more stable compared to the pure chemical compound. Blood analysis has shown that rosmarinic acid is a marker of the use of lemon balm in plasma, however, this compound is found in the form of a sulfoglucuronide in the blood after 12 weeks of use. No statistically significant differences were found between the concentrations of the tested drugs before and after 12 weeks of investigation. There was no increase in the number of copies of the virus in the blood. The monitored hematological and immunological parameters did not show a statistically significant change in values. Within the monitored biochemical parameters, there were no statistically significant changes that can be associated with the use of lemon balm, with the exception of the studied group of efavirenz, where there was a statistically significant increase in alanine aminotransferases and aspartate aminotransferases, but within reference values. It was shown that after 12 weeks of application of lemon balm, there was a statistically significant reduction in oxidative stress parameters in the studied groups of efavirenz and darunavir; in addition, a statistically significant increase in glutathione-S-transferase activity was shown in the efavirenz study group. Regarding the parameters of anthropometry, an increase in body weight was shown in the examined group of efavirenz after 12 weeks of application of lemon balm. The research presented here shows that the simultaneous use of lemon balm with antiretroviral therapy that includes one of the tested drugs does not affect the concentrations of these drugs. The monitored parameters in the patients showed that the use of lemon balm for 12 weeks in a dose of 600 mg was safe for patients. The presented positive results indicate a beneficial effect on HIV-infected patients with metabolic syndrome.

The aim of this study was to determine the levels of lipid peroxidation (MDA) and antioxidants such as reduced glutathione (GSH), catalase (CAT) and superoxide dismutase (SOD) in the blood serum of patients with cirrhosis and liver transplantation.In this study, serum malondialdehyde acid (MDA) levels, superoxide dismutase (SOD), reduced glutathione (GSH), and catalase (CAT) activities were measured spectrophotometrically and compared to the results of the healthy control group.SOD, CAT and GSH activities were significantly decreased in the patient groups compared to the healthy control group (p0.05). MDA levels were significantly higher in the patient group compared to the healthy control group (p0.05).In conclusion, this study demonstrated that oxidative stress may play an important role in the development of liver cirrhosis and in liver transplantation. This study is the first one to show how MDA, SOD, CAT and GSH levels change in liver cirrhosis and liver transplantation, while further studies are essential to investigate antioxidant enzymes and oxidative stress status in patients with cirrhosis and liver transplantation.Cilj ovog rada je bio da se odrede nivoi lipidne peroksidacije (MDA) i antioksidanti kao što su redukovani glutation (GSH), katalaza (CAT) i superoksid dismutaza (SOD) u krvi seruma pacijenata sa cirozom i transplantacijom jetre.U ovom izučavanju nivoi serumskog malondialdehida (MDA), superoksid dismutaze (SOD), redukovanog glutationa (GSH) i aktivnost katalaze (CAT) mereni su spektrofotometrijski i pore|eni sa rezultatima dobijenim kod zdrave kontrolne grupe.Vrednosti SOD, CAT i GSH bile su značajno niže u grupi zdravih (p0,05). Nivoi MDA bili su značajno viši u grupi pacijenata u odnosu na grupu zdravih osoba (p0,05).Na osnovu ovog izučavanja može se zaključiti da oksidativni stres ima značajnu ulogu u razvoju ciroze i transplantacije jetre. Ovo je prvo izučavanje koje ukazuje kako se nivoi MDA, SOD, CAT i GSH menjaju u cirozi jetre i transplantaciji jetre. Sledeća izučavanja biće značajna za proučavanje antioksidativnih enzima i statusa oksidativnog stresa u pacijenata sa cirozom i transplantacijom jetre.

Novija istraživanja ukazuju da svaka izloženost olovu, pa čak i veoma niskim dozama može biti štetna. Podaci o toksičnim mehanizmima i efektima olova iz dosadašnjih animalnih i humanih studija se uglavnom zasnivaju na izloženosti visokim dozama, te stoga postoji potreba da se ispitaju mehanizmi toksičnog dejstva i da se utvrde toksični efekti u uslovima produžene izloženosti niskim dozama olova. Imajući navedeno u vidu, cilj ove disertacije bio je da se na modelu subakutne izloženosti pacova ispita uticaj niskih doza olova na različite organe i sisteme organa. Studija je sprovedena na animalnom modelu Wistar pacova koji su podeljeni u 7 grupa po 6 jedinki od kojih je jedna grupa bila kontrolna a 6 ostalih grupa je tretirano tokom 28 dana rastućim dozama olova 0,1; 0,5; 1; 3; 7; 15 mg Pb/kg t.m./dan. Nakon 24 h od poslednje doze, pacovi su žrtvovani na human način a krv i organi su uzeti na dalju analizu. U krvi su određivani hematološki parametri, biohemijski parametri, hormoni, parametri oksidativnog statusa, bioelementi i olovo, dok su organi podvrgnti patohistološkoj analizi i u njima su određivani parametri oksidativnos stausa, bioelementi, olovo i aktivnost enzima acetilholinesteraze. Primena olova u šest niskih rastućih doza omogućila je modelovanje odnosa doza-odgovor i dobijanje Benchmark doza za ispitivane toksične efekte. Rezultati su pokazali da olovo pri niskim dozama, može ispoljiti toksične efekte skoro na svim ispitivanim organima. Dobijeni rezultati daju uvid u distribuciju olova između krvi i tkiva kao i internim dozama olova koje dovode do štetnih efekata. Izmenjeni profil hematoloških i biohemijskih parametara, zajedno sa izmenjenim nivoima bioelemenata, nastalim oksidativnim stresom, izmenjenom aktivnošću acetilholinesteraze i patohistološkom analizom ispitivanih tkiva, ukazuju na toksične efekte nakon subakutne oralne ekspozicije olovu. Najveći naučni doprinos ove doktorske disertacije je ispitivanje zavisnosti doza–odgovor za svaki pojedinačni parametar kao i određivanje Benchmark doze, koja će dalje doprineti sigurnijoj proceni rizika po zdravlje ljudi pri izloženosti niskim dozama olova. Najniža Benchmark doza dobijena u studiji je za efekat smanjenja nivoa testosterona u serumu pacova što predstavlja kritični toksični efekat studije. Sledeći efekti jesu inhibicija SOD u bubrezima, povećanje nivoa Cu u femuru, sniženje Cu u krvi i povećanje MDA u srcu, a zatim, povećanje TOS u mozgu, povećanje Zn u pankreasu i sniženje Cu u jetri. Recent research indicates that exposure to very low lead doses can be harmful. Data on the toxic mechanisms and effects of lead from previous animal and human studies are mainly based on exposure to high doses, and therefore there is a need to investigate the toxic mechanism and to determine toxic effects under conditions of prolonged exposure to low lead doses. Having this in mind, the aim of this dissertation was to examine the effects of low lead doses on various organs and organ systems in a subacute exposure rat model. The study was conducted on an animal model of Wistar rat. The rats were divided into 7 groups, with 6 rats in each, where one of the them was a control group. The rats from 6 other groups were treated for 28 days with increasing doses of 0.1; 0.5; 1; 3; 7; 15 mg Pb /kg b.w./day. After 24 hours from the last dose, rats were sacrificed and blood and organs were taken away for further analysis. Haematological parameters, biochemical parameters, hormones, oxidative status parameters, bioelements, and lead were determined in the blood, while the organs were subjected to pathohistological analysis and determination of oxidative status parameters, bioelements, lead and acetylcholinesterase enzyme activity. The use of lead in six low-increasing doses enable the modelling of the dose-response relationship and the obtaining of the Benchmark dose for the tested toxic effects. The results showed that lead at low doses can have toxic effects on almost all examined organs. The obtained results provide insight into the distribution of the lead between blood and tissues as well as the internal doses of lead that lead to harmful effects. Altered profiles of hematological and biochemical parameters, together with altered levels of bioelements, oxidative stress, altered acetylcholinesterase activity, and pathohistological analysis of examined tissues, indicate adverse effects after subacute lead exposure. The greatest scientific contribution of this doctoral dissertation is the examination of dose-response relationships for each individual parameter as well as the determination of the Benchmark dose, which will further contribute to a safer assessment of human health risk of low lead dose exposure. The lowest Benchmark dose obtained in the study was for the effect of reducing testosterone levels in rat serum, which is a critical toxic effect of the study. The next determined effects are inhibition of SOD in the kidneys, increased levels of Cu in the femur, decreased Cu in the blood and increased MDA levels in the heart, then, an increase in TOS in the brain, an increase in Zn in the pancreas and a decrease in Cu in the liver.

Parkinsonova bolest je najčešći neurodegenerativni poremećaj kretanja kojeg karakteriziraju progresija bolesti te kompleksna klinička slika s motoričkim i ne-motoričkim simptomima. Neurosteroidi dehidroepiandrosteron (DHEA) i dehidroepiandrosteron sulfat (DHEAS) te moždani neurotrofni čimbenik (BDNF) ključni su za neuroprotekciju, neurogenezu, memoriju i učenje te djeluju antioksidativno i protuupalno. Uzimajući u obzir ove činjenice, kao i činjenicu da trenutno ne postoji lijek za Parkinsonovu bolest, ističe se važnost istraživanja novih potencijalnih terapeutika poput DHEA, DHEAS i BDNF koji će liječenje učiniti učinkovitijim. Cilj ovog rada je istražiti potencijalne učinke DHEA, DHEAS i BDNF u prevenciji smrti dopaminergičkih neurona uporabom in vitro modela, odnosno staničnih linija ozlijeđenih neurotoksinima rotenonom i 6-hidroksidopaminom koji uzrokuju oštećenja stanica nalik onima u Parkinsonovoj bolesti. U tu svrhu, provedene su analize vijabilnosti stanica, parametara oksidativnog stresa te detekcija stanične smrti. Rezultati istraživanja upućuju na to da DHEA, DHEAS i BDNF pokazuju značajan neuroprotektivni potencijal što se očituje kroz povećanje preživljavanja stanica te smanjenje razine unutarstaničnih reaktivnih kisikovih jedinki i apoptotičnih, odnosno nekrotičnih stanica. Parkinson's disease is the most common neurodegenerative movement disorder characterized by disease progression and a complex clinical picture with motor and non-motor symptoms. The neurosteroids dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) and brain-derived neurotrophic factor (BDNF) are crucial for neuroprotection, neurogenesis, memory and learning, and have antioxidant and anti-inflammatory effects. Considering these facts, as well as the fact that there is currently no cure for Parkinson's disease, the importance of research for new potential therapeutics such as DHEA, DHEAS and BDNF that will make the treatment more effective is essential. The aim of this thesis is to investigate the potential effects of DHEA, DHEAS and BDNF in preventing the death of dopaminergic neurons using in vitro model, i.e. cell lines injured by the neurotoxins rotenone and 6- hydroxydopamine, which cause cell damage similar to those in Parkinson's disease. For this purpose, analyzes of cell viability, oxidative stress parameters and detection of cell death were carried out. Research results indicate that DHEA, DHEAS and BDNF show a significant neuroprotective potential, which is manifested through an increase in cell survival and a decrease in the level of intracellular reactive oxygen species and apoptotic and/or necrotic cells.

Oksidativni stres se javlja zbog povećane količine reaktivnih kisikovih spojeva (ROS) ili smanjenog kapaciteta enzimatskih i neenzimatskih antioksidativnih sustava. Alfa lipoična kiselina je moćan antioksidans s trenutnom primjenom u terapiji dijabetičke polineuropatije. Brojna istraživanja proučavaju utjecaj alfa lipoične kiseline na oksidativni stres i njenu potencijalnu primjenu u terapiji raznih bolesti povezanih s oksidativnim stresom. Cilj ovog rada bio je ispitati utjecaj suplementacije alfa lipoičnom kiselinom na ukupan antioksidativni kapacitet seruma, a s obzirom na neke parametre životnog stila pacijentica (pretilost, pušenje, korištenje dodataka prehrani i način prehrane). Antioksidativni status pacijentica određen je mjerenjem reduktivnog potencijala plazme i mjerenjem aktivnosti superoksid dismutaze (SOD). Nije uočen značajni utjecaj pušenja, korištenja dodataka prehrani, indeksa tjelesne mase niti načina prehrane na aktivnost SOD. Podskupina pacijentica s najvišim unosom povrća (1.kvartil) imale su značajno veći reduktivni potencijal plazme od ostalih pacijentica. Suplementacija sa 600mg alfa-lipoične kiseline/dne kroz 3 mjeseca nije značajno utjecala na reduktivni potencijal plazme niti aktivnost SOD u krvi ispitanica. Potrebno je provesti dodatna istraživanja s većim brojem ispitanika, različitim biomarkerima oksidativnog stresa i različitim dozama alfa lipoične kiseline da bi se utvrdio utjecaj suplementacije alfa lipoične kiseline na antioksidativni kapacitet. Oxidative stress may occur as a result of an increased amount of reactive oxygen species (ROS) or reduced enzymatic or nonenzymatic antioxidant system capacity. Alpha lipoic acid is a potent antioxidant, currently used in the treatment of diabetic polyneuropathy. Numerous trials are examining the effect of alpha lipoic acid on oxidative stress and its potential application in the treatment of various oxidative stress related diseases. The goal of this paper was to examine the effect of alpha lipoic acid supplementation on total serum antioxidant capacity, in regard to lifestyle parameters (obesity, smoking, diet and dietary supplements). Antioxidative status of the patients was determined using ferric reducing ability of plasma assay and by measuring superoxide dismutase activity (SOD). There was no significant observed effect of smoking, use of dietary supplements, body mass index, or diet on SOD activity. The group of patients with the highest vegetable consumption (1st quartile) showed significantly higher reducing ability of plasma compared to other patients. 90 day 600mg/day alpha lipoic acid supplementation showed no significant effect on reducing ability of plasma or SOD activity in the blood of the patients. Additional research should be conducted using a larger number of participants, different oxidative stress biomarkers and different doses of alpha lipoic acid to determine the effects of alpha lipoic acid supplementation on antioxidant capacity.

Podlogu brojnih kroničnih bolesti čini oksidativni stres, odnosno stanje neravnoteže između stvaranja slobodnih radikala (oksidansa) i antioksidativne obrane organizma u korist oksidansa. Stoga je adekvatan unos egzogenih antioksidansa važan doprinos prevenciji i/ili liječenju bolesti povezanih s oksidativnim stresom. α-lipoična kiselina (ALA) je zbog svojih višestrukih bioloških uloga (neutralizacija slobodnih radikala, regeneracija endogenih antioksidansa, popravak oksidiranih oštećenja, kelacija metala, itd.) i dobre raspodjele u organizmu moćan antioksidans. Cilj ovog diplomskog rada bio je ispitati utjecaj suplementacije α-lipoičnom kiselinom na ukupni antioksidativni potencijal seruma pacijentica s dijagnozom cervikalne intraepitelne neuroplazije. Istraživanje je provedeno kao dvostruko-slijepa placebom kontrolirana studija u kojoj su pacijentice kroz period od 3 mjeseca dnevno suplementirane sa 600 mg ALA. Modificiranom ORAC metodom izmjeren je antioksidativni potencijal seruma te uspoređen utjecaj određenih parametara životnog stila i prehrambenih navika pacijentica. Uočeno je da unos voća i povrća (3 ili više porcija dnevno) prehranom značajno povećava antioksidativni potencijal seruma pacijentica. Suprotno tome, nije uočena razlika antioksidativnog potencijala seruma pacijentica s obzirom na pušenje i indeks tjelesne mase. Suplementacija α-lipoičnom kiselinom nije značajno utjecala na vrijednosti ORAC antioksidativnog potencijala. Trenutno je dostupan vrlo ograničen broj znanstvenih studija koje pokazuju oprečne rezultate vezano uz učinkovitost ALA u prevenciji oksidativnog stresa te je u tom kontekstu potrebno provesti dodatna istraživanja. The basis of many chronic diseases is oxidative stress; that is, a state of imbalance between the creation of free radicals (oxidants) and the body's antioxidant defense, in favor of oxidants. Therefore, optimizing the intake of antioxidans is crucial in the context of prevention and/or treatment of oxidative stress-related diseases. α-lipoic acid (ALA) is a powerful antioxidant due to its multiple biological roles (neutralization of free radicals, regeneration of endogenous antioxidants, repair of oxidized damage, metal chelation, etc.) and adequate distribution in the body. The aim of this thesis was to examine the influence of supplementation with α-lipoic acid on the total antioxidant potential in the serum of patients diagnosed with cervical intraepithelial neuroplasia. The research was conducted as a double-blind placebo-controlled study in which patients were supplemented with 600 mg of ALA daily for a period of 3 months. Using the modified ORAC method, the antioxidant potential of serum was measured and the influence of certain parameters of the lifestyle and eating habits of the patients was compared. It was observed that the intake of fruits and vegetables (3 or more portions per day) significantly increased the antioxidant potential of female patients. On the contrary, no difference was observed in the antioxidant potential of female patients related to smoking and body mass index. Supplementation with α-lipoic acid did not significantly affect the ORAC serum values. The effectiveness of ALA in the prevention of oxidative stress has not been properly investigated and the number of available studies is limited. Therefore, further research is needed in order to get better insight into antioxidant efficiency of ALA.

Kronična vnetna črevesna bolezen (KVČB) je bolezen prebavil, pri kateri se izmenjavajo aktivne faze in obdobja remisije. Mehanizem nastanka še ni znan, zato na tem področju potekajo številne raziskave, ki so prinesle boljše poznavanje patogeneze in zdravljenja, na področju diagnostike pa je še prostor za izboljšave. Zlati diagnostični standard ostaja endoskopija, ki je zaradi invazivnosti neprijetna za bolnike. Študije kažejo, da oksidativni stres prispeva k razvoju in napredovanju KVČB, zato smo se v magistrski nalogi odločili preučiti, s katerim kazalnikom oksidativnega stresa bi lahko razlikovali med aktivno fazo bolezni in remisijo. V ta namen smo naredili sistematični pregled literature in meta-analizo. V sistematični pregled literature smo vključili 59 člankov na osnovi vključitvenih in izključitvenih kriterijev. Ocenili smo pristranost v meta-analizi uporabljenih 46 člankov. Rezultati so pokazali, da ima 26 raziskav nizko, 10 raziskav srednje in 10 raziskav visoko tveganje za pristranost. Meta-analizo smo izvedli v programu Review Manager 5.4., rezultate prikazali v obliki drevesnih diagramov, velikost učinka pa izrazili kot standardizirano povprečno razliko (SMD) med bolniki in zdravimi. Glede na zdrave so bili statistično značilno povišani (SMD): napredni oksidirani proteinski produkti (AOPP) (1,81), malonildialdehid in s tiobarbiturno kislino reagirajoče snovi (0,87) ter glutation peroksidaza (0,67). Kazalniki, ki so bili glede na zdrave statistično značilno znižani so: glutation (-1,56), celokupni status antioksidantov in celokupna kapaciteta antioksidantov (-1,32), albumin (-1,29), bilirubin (-1,09), vitamin A (-0,86), transferin (-0,75), prosti tioli (-0,69), vitamin C (-0,66) ter vitamin E (-0,41). Dodatno so bili bilirubin (v aktivni fazi -3,10, v remisiji -0,78), katalaza (CAT) (v aktivni fazi -0,68, v remisiji -0,10) in transferin (v aktivni fazi -1,17, v remisiji -0,33) statistično pomembno znižani glede na SMD pri bolnikih v aktivni fazi in remisiji. Vrednost SMD za AOPP pa je bila pri aktivni fazi (3,59) statistično pomembno povišana glede na remisijo (0,89). Pri izvedbi občutljivostne analize z izključitvijo raziskav z visokim tveganjem za pristranost nismo opazili statistično značilne spremembe velikosti učinka za noben kazalnik. Potrebne bodo nadaljnje študije, ki bodo pokazale, kako točno je oksidativni stres vpleten v KVČB. Meritve koncentracij oz. aktivnosti bilirubina, AOPP, CAT in transferina bi lahko pripomogle k obvladovanju in diagnostiki KVČB, saj lahko z njimi hitreje in manj invazivno zaznavamo vnetne procese. Inflammatory bowel disease (IBD) is a gastrointestinal disease, characterized by episodes of active and inactive phases. Many studies are ongoing as the mechanism of disease progression is still unknown. Studies contributed to better knowledge of pathogenesis and treatment but there is still space for improvements in diagnostics. Endoscopy is the main diagnostic standard which is unpleasant for patients due to its invasiveness. Studies show that oxidative stress contributes to the development and progression of IBD, therefore the goal of this master’s thesis was to determine markers of oxidative stress which could help distinguish between active and inactive phase of IBD. For this purpose, a systematic literature review and meta-analysis were carried out. Systematic review included 59 articles based on inclusion and exclusion criteria. The 46 articles included in the meta-analysis were assessed for risk of bias. 26 articles were of good, 10 articles of fair and 10 articles of poor quality in terms of risk of bias. Meta-analysis was carried out with Review Manager 5.4., results were presented through forest plots and the size effect were in form of standardized mean difference (SMD) between patients and healthy controls. Concentration or activity was significant increased by IBD patients compared to healthy controls for next markers (SMD): advanced oxidation protein products (AOPP) (1,81), malondialdehyde and thiobarbituric acid reactive substances (0,87) and glutathione peroxidase (0,67). Concentration or activity was significant decreased by IBD patients compared to healthy controls for glutathione (-1,56), total antioxidant capacity and total antioxidant status (-1,32), albumin (-1,29), bilirubin (-1,09), vitamin A (-0,86), transferrin (-0,75), free thiols (-0,69), vitamin C (-0,66) and vitamin E (-0,41). Additionally, were bilirubin (in active phase -3,10, in inactive phase -0,78), catalase (CAT) (in active phase -0,68, in inactive phase -0,10) and transferrin (in active phase -1,17, in inactive phase -0,33) significant decreased according to SMD by active compared to inactive IBD. SMD value for AOPP was statistically increased by active (3,59) compared to inactive IBD (0,89). Using a sensitivity analysis with excluded high risk of bias articles we did not observe a statistically relevant change in the overall effect for any of the markers. Further studies are needed to point out how oxidative stress is involved in IBD and its contribution to pathogenesis. Measurements of concentration or activity of bilirubin, CAT, transferrin and AOPP could contribute to control and diagnostics of IBD as measurements of markers are faster and less invasive detection of inflammatory processes.

Oxidation is the second most common degradation pathway for pharmaceuticals, after hydrolysis. However, in contrast to hydrolysis, oxidation is mechanistically more complex and produces a wider range of degradation products oxidation is thus harder to control. The propensity of a drug towards oxidation is established during forced degradation studies. However, a more realistic insight into degradation in the solid state can be achieved with accelerated studies of mixtures of drugs and excipients, as the excipients are the most common sources of impurities that have the potential to initiate oxidation of a solid drug product. Based on the results of these studies, critical parameters can be identified and appropriate measures can be taken to avoid the problems that oxidation poses to the quality of a drug product. This article reviews the most common types of oxidation mechanisms, possible sources of reactive oxygen species, and how to minimize the oxidation of a solid drug product based on a well-planned accelerated study.

Poznato je kako je kod bolesnika liječenih HD-om poremećaji statusa uhranjenosti i OS-a značajno doprinose ukupnoj i srčano-žilnoj smrtnosti koja je kod ove skupine bolesnika značajno viša nego u općoj populaciji. Cilj ovog istraživanja je bio istražiti promjenjivost pokazatelja statusa uhranjenosti i OS-a te njihovu međusobnu povezanost tijekom šestomjesečnog praćenja. U istraživanje je uključeno 45 bolesnika, prosječne dobi 70 godina (raspon 60,5-76,5 godina) liječenih HD-om prosječno 27 mjeseci (16-53,5 mjeseci). Mjerenja antropometrijskih i biokemijskih pokazatelja statusa uhranjenosti, analiza sastava tijela sa BIS-om te procjena statusa uhranjenosti standardiziranim upitnicima za bolesnike liječene HD-om provedeni su u tri puta tijekom 6 mjeseci (lipanj, listopad, siječanj). Nadalje, za procjenu OS-a korišteni su testovi oksidativnog oštećenja molekula poput reaktivnih kisikovih metabolita (d-ROMs) i lipidne peroksidacije (TBARS), ali i pokazatelji antioksidativne obrane organizma poput sadržaja tiola na proteinima plazme (TIOLI) i antioksidativni kapacitet plazme (FRAP) te razina vitamina C. Također ispitano je postojanje utjecaja doze dijalize (mjereno sa Kt/V i URR) i doze EPO-a na statusa uhranjenosti i OS. Rezultati su pokazali kako je tijekom šestomjesečnog praćenja došlo do značajnog povišenja pokazatelja masnog tkiva: FTI (p, It is well known that in HD patients disorders of nutritional status and OS significantly contribute to all-cause and cardiovascular mortality in HD patients which is proven to be significantly higher in this group of patients than in general population. Aim of this study was to assess variability of markers of nutritional status and biomarkers of OS and their possible interactions during six months follow-up. Study included 45 patients, 70 years of age (60.5 -76.5 years) with median dialysis duration of 25 months (16-53.5 months). During follow-up we measured three times (June, October and Januar) anthropometric and biochemical parameters of nutritional status, body composition analysis and assessment of nutritional status using MIS and DMS nutritional questionnaire for HD patients. Further, OS analysis was performed by using tests showing oxidative damage like reactive oxygen metabolites (d-ROMs) and products of lipid peroxidation (TBARS) together with tests for assessing antioxidative defence like protein plasma reduced thiol content (THIOLS) and antioxidation capacity of plasma (FRAP) with levels of vitamin C. Also, study assessed influence of dialysis dose (measured with Kt/V and URR) and EPO dose on nutritional status and OS. Results showed that during follow-up there was a significant increase of body composition markers that reflect adipose tissue: FTI (P

Oxidative stress is associated with a wide range of diseases characterised by oxidant-mediated disturbances of various signalling pathways and cellular damage. The only effective strategy for the prevention of cellular damage is to limit the production of oxidants and support their efficient removal. The implication of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in the cellular redox status has spurred new interest in the use of its natural modulators (e.g., curcumin, resveratrol). Unfortunately, most natural Nrf2 modulators are poorly soluble and show extensive pre-systemic metabolism, low oral bioavailability, and rapid elimination, which necessitates formulation strategies to circumvent these limitations. This paper provides a brief introduction on the cellular and molecular mechanisms involved in Nrf2 modulation and an overview of commonly studied formulations for the improvement of oral bioavailability and in vivo pharmacokinetics of Nrf2 modulators. Some formulations that have also been studied in vivo are discussed, including solid dispersions, self-microemulsifying drug delivery systems, and nanotechnology approaches, such as polymeric and solid lipid nanoparticles, nanocrystals, and micelles. Lastly, brief considerations of nano drug delivery systems for the delivery of Nrf2 modulators to the brain, are provided. The literature reviewed shows that the formulations discussed can provide various improvements to the bioavailability and pharmacokinetics of natural Nrf2 modulators. This has been demonstrated in animal models and clinical studies, thereby increasing the potential for the translation of natural Nrf2 modulators into clinical practice.

The treatment with fixed orthodontic appliances could have an important role in the induction of oxidative stress and associated negative consequences. Because of the simultaneous effects of corrosion, deformation, friction, and mechanical stress on fixed orthodontic appliances during treatment, degradation of orthodontic brackets and archwires occurs, causing higher concentrations of metal ions in the oral cavity. Corroded appliances cause the release of metal ions, which may lead to the increased values of reactive oxygen species (ROS) due to metal-catalyzed free radical reactions. Chromium, iron, nickel, cobalt, titanium, and molybdenum all belong to the group of transition metals that can be subjected to redox reactions to form ROS. The estimation of health risk due to the amount of heavy metals released and the level of selected parameters of oxidative stress generated for the time of treatment with fixed orthodontic appliances is presented. Approaches to avoid oxidative stress and recommendations for the preventive use of topical or systemic antioxidants during orthodontic treatment are discussed.

Primary Sjögren's syndrome (pSS) is a disease associated with the overexpression of proinflammatory cytokines, and oxidative stress is one of the factors responsible for its etiopathogenesis. This study aimed to investigate the thiol/disulphide homeostasis in pSS patients.The study included 68 pSS patients and 69 healthy controls. Thiol/disulphide homeostasis (total thiol, native thiol, and disulphide levels) was measured using the automatic spectrophotometric method developed by Erel and Neselioglu, and the results of the 2 groups were compared.The gender and age distributions of the pSS and control groups were similar (It was concluded that the thiol/disulphide balance shifted towards disulphide in patients with pSS.Primarni Sjögrenov sindrom (pSS) je bolest povezana sa prekomernom ekspresijom proinflamatornih citokina, dok je oksidativni stres jedan od faktora odgovornih za njegovu etiopatogenezu. Cilj ove studije je bio da ispita tioldisulfidnu homeostazu kod pacijenata sa pSS-om.Studija je obuhvatila 68 pacijenata sa pSS-om i 69 zdravih kontrolnih pojedinaca. Homeostaza tiol-disulfida (nivoi ukupnog tiola, nativnog tiola i disulfida) je izmerena pomoću automatske spektrofotometrijske metode koju su razvili Erel i Neselioglu. Rezultati dve grupe su upoređeni.Raspodela po polu i starosti pacijenata sa pSSom i pojedinaca u kontrolnoj grupi je bila slična (Zaključeno je da se ravnoteža tiol/disulfid pomera prema disulfidu kod pacijenata sa pSS-om.

Oxidative stress and neuroinflammation are involved in the pathogenesis and progression of glaucoma. Our aim was to evaluate the impact of selected single-nucleotide polymorphisms in inflammation and oxidative stress genes on the risk of glaucoma, the patients’ clinical characteristics and the glaucoma phenotype. In total, 307 patients with primary open-angle glaucoma or ocular hypertension were enrolled. The control group included 339 healthy Slovenian blood donors. DNA was isolated from peripheral blood. Genotyping was performed for SOD2 rs4880, CAT rs1001179, GPX1 rs1050450, GSTP1 rs1695, GSTM1 gene deletion, GSTT1 gene deletion, IL1B rs1143623, IL1B rs16944, IL6 rs1800795 and TNF rs1800629. We found a nominally significant association of GSTM1 gene deletion with decreased risk of ocular hypertension and a protective role of IL1B rs16944 and IL6 rs1800629 in the risk of glaucoma. The CT and TT genotypes of GPX1 rs1050450 were significantly associated with advanced disease, lower intraocular pressure and a larger vertical cup–disc ratio. In conclusion, genetic variability in IL1B and IL6 may be associated with glaucoma risk, while GPX and TNF may be associated with the glaucoma phenotype. In the future, improved knowledge of these pathways has the potential for new strategies and personalised treatment of glaucoma.

Serum paraoxonase-1 (PON1) is the most studied member of the group of paraoxonases (PONs). This enzyme possesses three enzymatic activities: lactonase, arylesterase, and paraoxonase activity. PON1 and its isoforms play an important role in drug metabolism as well as in the prevention of cardiovascular and neurodegenerative diseases. Although all three members of the PON family have the same origin and very similar amino acid sequences, they have different functions and are found in different locations. PONs exhibit substrate promiscuity, and their true physiological substrates are still not known. However, possible substrates include homocysteine thiolactone, an analogue of natural quorum-sensing molecules, and the recently discovered derivatives of arachidonic acid—bioactive δ-lactones. Directed evolution, site-directed mutagenesis, and kinetic studies provide comprehensive insights into the active site and catalytic mechanism of PON1. However, there is still a whole world of mystery waiting to be discovered, which would elucidate the substrate promiscuity of a group of enzymes that are so similar in their evolution and sequence yet so distinct in their function.

Water activity (a$_w$) is critical for microbial growth, as it is severely restricted at a$_w$ < 0.90. Saturating NaCl concentrations (~5.0 M) induce extreme water deprivation (aw ≅ 0.75) and cellular stress responses. Halophilic fungi have cellular adaptations that enable osmotic balance and ionic/oxidative stress prevention to grow at high salinity. Here we studied the morphology, osmolyte synthesis, and oxidative stress defenses of the halophile Aspergillus sydowii EXF-12860 at 1.0 M and 5.13 M NaCl. Colony growth, pigmentation, exudate, and spore production were inhibited at NaCl-saturated media. Additionally, hyphae showed unpolarized growth, lower diameter, and increased septation, multicellularity and branching compared to optimal NaCl concentration. Trehalose, mannitol, arabitol, erythritol, and glycerol were produced in the presence of both 1.0 M and 5.13 M NaCl. Exposing A. sydowii cells to 5.13 M NaCl resulted in oxidative stress evidenced by an increase in antioxidant enzymes and lipid peroxidation biomarkers. Also, genes involved in cellular antioxidant defense systems were upregulated. This is the most comprehensive study that investigates the micromorphology and the adaptative cellular response of different non-enzymatic and enzymatic oxidative stress biomarkers in halophilic filamentous fungi.

Kaspaze so živalskecisteinske proteaze, ki igrajo odločilno vlogo pri regulaciji in izvedbi programirane celične smrti. Kaspaz pri neživalskih evkariontih ni, lahko pa tam najdemo njihove strukturne homologe, metakaspaze. Te ločimo na tri tipe, ki se med seboj razlikujejo po strukturi. Njihova natančna funkcija ni znana, je pa verjetno med drugim povezana tudi z odzivom na oksidativni stres. V sklopu magistrskega dela smo se osredotočili na metakaspazo tipa I iz enocelične zelene alge Chlamydomonas reinhardtii, CrMCA-I. Piprava divjega tipa te proteaze v E. coli ni bila uspešna, saj se je ta proteaza sprva agregirala v netopni frakciji, dokler ji nismo odstranili hidrofobne N-končne regije in zanke 360, ki je značilna za metakaspaze tipa I iz organizmov iz klada Viridiplantae. Tako modificiran CrMCA-I (CrMCA-I_CL) smo izrazili v E. coli in ga izolirali. Podobno smo modificirali tudi zapisa za dve metakaspazi tipa I iz modelne rastline Arabidopsis thaliana, AtMCA-Ia in AtMCA Ib, vendar nam jih ni uspelo pridobiti v topni frakciji. Izoliranemu CrMCA-I_CL smo določili aktivnost pri različnih koncentracijah kalcijevih ionov in pri različnih pH. Ugotovili smo, da je CrMCA I_CL neaktiven v odsotnosti Ca2+, največjo aktivnost pa doseže v nizkih milimolarnih koncentracijah Ca2+. Njegov pH optimum je v nevtralnem. Poleg tega smo poskusili nemodificirano obliko CrMCA-I izraziti tudi v C. reinhardtii. Pri tem smo naleteli na težave z vzpostavljanjem stabilnih kolonij po transformaciji celic. Uspelo nam je pridobiti kolonije na trdnem gojišču z antibiotikom, vendar te kolonije po inokulaciji v tekoče gojišče niso zrasle. Da bi ugotovili, ali lahko s protitelesi, ki so bila ustvarjena proti metakaspazi GtMCA-III, spremljamo prisotnost metakaspaz pri odzivu na oksidativni stres, smo pri divjem tipu C. reinhardtii in pri insercijski mutanti C. reinhardtii CrMCA I::AphVII inducirali oksidativni stres. Glede na to, da metakaspaz s protitelesi v vzorcih nismo zaznali, bomo v prihodnje morali za njihovo prisotnost in aktivnost pri oksidativnem stresu uporabiti drugačne metode. Caspases are cysteine proteases in animals that play an important role in programmed cell death regulation and execution. Caspases are absent in non animal eucaryotes however, they contain structural homologues of caspases, metacaspases. These can be divided into three types that can be differentiated based on their domain architectures. Their function is yet not known, although it is likely linked to oxidative stress responses. In this work, we focused on CrMCA-I, the only type I metacaspase from the unicellular green alga Chlamydomonas reinhardtii. When expressed in E. coli, it aggregated in the insoluble fraction. However, removal of the N-terminal region and the 360 loop (CrMCA-I_CL) resulted in expression of soluble and proteolytically active protease. Since this loop is characteristic of type I metacaspases from organisms of the Viridiplantae clade, we also tried to express two sequences encoding metacaspases from Arabidopsis thaliana, AtMCA-Ia and AtMCA-Ib. However, we were not successful in their expression. We measured the activity of the isolated CrMCA-I_CL at different concentrations of calcium ions and different pH values. CrMCA-I_CL was shown to be inactive in the absence of Ca2+ ions and reached its maximum activity at low millimolar Ca2+ concentrations. Its pH optimum was at neutral values. We also tried to express non-modified CrMCA-I homologously in C. reinhardtii, but we encountered problems with establishing stable colonies after transformation. While colonies did appear on solid medium with antibiotic, no growth could be observed in liquid media. To asses if presence of metacaspases during oxidative stress response could be monitored using anti-metacaspase antibodies, we induced oxidative stress in the wild type C. reinhardtii and in CrMCA-I insertion mutant CrMCA-I::AphVII. Since no metacaspase could be detected, other methods will have to be used in the future for monitoring the presence and activity of metacaspases in this organism.

Na tržištu danas postoje različiti materijali koji se upotrebljavaju pri proizvodnji fiksnih ortodontskih naprava od kojih je najčešća “edgewise”, naprava, koja se sastoje od lukova, ligatura i bravica, za čiju je adheziju na zub potreban kompozitni materijal. Sama terapija obično se procjenjuje na period trajanja od 24 mjeseci, te se tokom čitavog tog perioda ortodontske bravice, lukovi, ligature i kompozit nalaze u usnoj šupljini izlažući se procesima degradacije i mogućoj sposobnosti generiranja reaktivnih kisikovih spojeva (ROS) vodeći oštećenju staničnih elemenata, smanjenju stanične proliferacije i diferencijacije te apoptozi. Cilj istraživanja bio je ispitati utjecaj dentalnih materijala na metaboličku aktivnost dviju vrsta kvasaca Saccharomyces cerevisiae: W303 i divlji tip, te procijeniti njihov citotoksični učinak u odnosu na: vrijeme izloženosti (3, 7, 14 i 28 dana), vrsti kvasca u umjetnoj slini ili YPD hranidbenoj podlozi, te prisustvu ortodontskih materijala (bravica i lukova). U praćenju metaboličke aktivnosti koristili su se testovi stanične proliferacije (XTT test) i “Live/Dead Yeast Viability kit”, te AO/PI boja za određivanje vijabilnosti stanica čiji su rezultati prikazani krivuljama rasta i putem prebrojanih stanica na LUNA-FLTM uređaju. Tretmani ortodontskim bravicama, lukovima i kompozitom utječu na metaboličku aktivnost i vijabilnost stanica kvasca te usporavaju rast stanica i njihovu diobu.

Onečišćenje mikroplastikom predstavlja globalni problem, posebno zbog njezine akumulacije iz okoliša u organizme, što dovodi do brojnih negativnih učinaka, uključujući oksidativni stres. Prisutnost mikroplastike u riječnim ekosustavima i njezin utjecaj na slatkovodne organizme općenito su slabo istraženi. Ipak, u Hrvatskoj je zagađenje mikroplastikom potvrđeno u estuariju rijeke Krke, nizvodno od Nacionalnog parka „Krka“. Budući da je područje uzvodno od granice Parka pod pritiskom industrijskih i komunalnih otpadnih voda grada Knina, cilj istraživanja bio je prikupiti podatke o prisutnosti mikroplastike u uzvodnom dijelu rijeke Krke i unosu u dominantnu vrstu riba, potočnu pastrvu (Salmo trutta, Linnaeus 1758). Uzorkovanje riba provedeno je u proljeće i jesen 2021. na tri lokacije: izvor rijeke Krke (referentna postaja, KRS), nizvodno od komunalnog ispusta grada Knina (KRK) i unutar nacionalnog parka (KBL), a riječna voda je dodatno uzorkovana na postaji s industrijskom otpadnom vodom (IWW) i na dvjema pritokama rijeke Krke, Butišnici (TBU) i Orašnici (TOR). U svim je uzorcima vode (nakon filtriranja) i sadržaja probavila riba (nakon razgradnje s HNO3 i HF) utvrđena prisutnost fragmenata mikroplastike pregledavanjem pod lupom. Koncentracija glutationa (GSH), biomarkera antioksidativnog kapaciteta, izmjerena je spektrofotometrijski u mišiću i probavilu riba. Porast GSH nizvodno od izvora u obje sezone ukazuje na mogući oksidativni stres u organizmima izloženima otpadnim vodama. Najveća razlika između postaja zabilježena je u tkivu mišića u proljeće, kada je razina GSH na KBL bila 1, 4 puta veća nego na KRS. Prosječne koncentracije bile su više u probavilu (1100 – 1542 nmol/g tkiva) nego u mišiću (242 - 338 nmol/g tkiva), a sezonske razlike su zabilježene samo na nizvodnim postajama, s nižim koncentracijama izmjerenim u jesen. Zbog potvrđene prisutnosti mikroplastike u riječnoj vodi, kao i njezinog unosa u pastrve putem hrane i povećanog antioksidativnog odgovora nizvodno od ispusta otpadnih voda potrebno je nastaviti istraživanja prisutnosti i bioloških učinaka mikroplastike u rijeci Krki.

donosne pčele (Apis mellifera). Izaziva bolest nozemozu (nozematozu) i u zavisnosti od stepena infekcije i jačine pčelinjih društava, ostavlja značajne posledice na zdravlje, reproduktivne i produktivne sposobnosti pčela. Antibiotik fumagilin je godinama bio jedini lek dostupan za kontrolu ovog patogena. Njegova primena dovedena je u pitanje zbog nedostataka koji prate njegovu primenu (efikasnost, mortalitet pčela i pojava rezidua u pčelinjim proizvodima). Širom sveta se sprovode istraživanja sa ciljem pronalaska alternative za kontrolu nozeme. U ovom radu prikazani su naši rezultati ispitivanja dijetetskih suplemenata na bazi prirodnih/biljnih ekstrakata (Medenko forte i ekstrakti gljiva Agaricus blazei i A. bisporus), timola i vitaminsko-mineralnog aditiva (BEEWELL AminoPlus). Kod pčela tretiranih svim nabrojanim suplementima i fumagilinom kao kontrolnim tretmanom, praćen je broj spora N. ceranae, nivoi ekspresije gena značajnih za imunitet pčela kao i parametri oksidativnog stresa. Svi preparati primenjivani su preventivno (tri dana pre infekcije), u trenutku inficiranja, kao i tri dana nakon infekcije sa N. ceranae, u cilju određivanja najboljeg momenta za primenu. Dokazani su negativni efekti fumagilina kada je preventivno primenjivan kod neinficiranih pčela. Kod inficiranih pčela većina testiranih suplemenata dovela je do smanjenja broja spora N. ceranae, a pokazali su i zaštitni efekat u smislu sprečavanja imunosupresije i oksidativnog stresa. The microsporidian Nosema ceranae is an obligate intestinal parasite of the honey bee (Apis mellifera). It causes nosemosis and, depending on the infection level and the strength of the infected bee colonies, causes significant health disorders, including malfunction of reproductive and productive capabilities of bees. The antibiotic fumagillin was the only drug available for Nosema control for many years. However, use of fumagillin has been questioned due to the drawbacks related to its efficacy, bee mortality and the presence of residues in bee products. Studies aimed to find alternatives for Nosema control are being conducted worldwide. In this paper, our results related to diet supplements based on natural/plant extracts (Medenko forte and extracts of Agaricus blazei and A. bisporus), thymol and vitamin-mineral additive (BEEWELL AminoPlus) are presented. Bees were treated with these supplements and fumagillin (control treatment) and gene expression levels (important for bee immunity), as well as oxidative stress parameters were assessed. In order to determine adequate time for application, all supplements were applied preventively (three days before infection), at the time of infection, as well as three days after N. ceranae infection. In uninfected bees, fumagillin demonstrated adverse effects when applied prophylactically. In Nosema-infected bees, tested supplements mainly contributed to the decrease of N. ceranae spore levels, but also exhibited a protective effect based on level of immune-related gene expression and parameters of oxidative stress.

U današnje vrijeme sve veća je osviještenost o malokluzijama i potrebom za nošenjem ortodontskih naprava. Najčešće korištena ortodontska naprava je fiksna naprava koji se sastoji od metalnih elemenata (bravica i žica), a za njihovu adheziju na zub koriste kompozitni materijali. Svi ovi elementi u svom sastavu imaju tvari koje mogu inducirati oksidativni stres te posljedično oštetiti stanične elemente, smanjiti njihovu metaboličku aktivnost, i naposlijetku dovesti do apoptoze ili nekroze stanica. Toksičnost se ispitivala na dva soja stanicama kvasca Saccharomyces cerevisiae - W303 i divlji tip, na dvije vrste podloge (kvaščeva podloga i umjetna slina), a korištene su dvije vrste kompozita (GC Ortho Connect i ConTec Go!), čelične bravice i NiTi lukovi. Rezultati pokazuju da kvasci otežano rastu u otopinama impregniranim ovim materijalima te da se dešavaju promjene na razini njihove morfologije i fiziologije. U stadijima povećanog oksidativnog stresa, mijenja se veličina stanice i grupiraju se u klastere od dvije ili više stanica. Vijabilnost ispitana AO/PI i FDA bojenjem se pokazala smanjena u otopinama s materijalima u odnosu na kontrolu, kao i metabolička aktivnost mjerena XTT testom. Zaključno slobodni radikali ispušteni iz ortodontskih materijala negativno utječu na vitalnost, proliferaciju i vijabilnost oba soja kvasaca. Stoga je potreban povećan oprez u korištenju ovih materijala, razmišljanje o manje štetnim alternativama (keramičke bravice, bisGMA-free kompoziti) ili uključivanje antioksidantnih preparata kod pacijenata u terapiji fiksnim ortodontskim napravama.

Oksidativni stres (OS) predstavlja poremećaj ravnoteže između antioksidanasa i prooksidanasa i pritom je posledica smanjenog sadržaja antioksidanasa ili povećanog nivoa reaktivnih kiseoničnih vrsta (ROS) u organizmu. Danas se smatra da OS ima značajnu ulogu u patogenezi velikog broja oboljenja, među kojima su i bolesti bubrega. Humani serumski albumin (HSA) pored brojnih fizioloških funkcija, ima ulogu u odbrani organizma od oksidativnog stresa. Prisustvo jedne slobodne tiolne grupe ostatka Cys34 na površini molekula HSA jeste glavni razlog ove bitne funkcije HSA. Primenom elektroforetskih i imunoblot metoda, dvostepenim izolovanjem HSA, UV/VIS analizom i spektrofluorimetrijom, dobijeni su eksperimentalni rezultati, čija je statistička analiza dala uvid u razlike strukturnih karakteristika ovog proteina kod analiziranih grupa pacijenata. Osnovni cilj ovog rada bilo je utvrđivanje antioksidativnog kapaciteta seruma (sa naglaskom na slobodne tiolne grupe) četiri različite grupe uzoraka: tri grupe pacijenata sa različitim stadijumima bolesti bubrega i različitim modalitetima lečenja i kontrolne grupe zdravih osoba. Primarni zadatak bilo je određivanje koncentracija slobodnih tiolnih grupa, kako ukupnih tako i onih na molekulima HSA, čiji je on glavni izvor u cirkulaciji. Ova saznanja su od velikog značaja jer se na taj način jasno mogu povezati različiti stadijumi bubrežne insuficijencije i/ili različiti tretmani (hemodijaliza i peritonealna dijaliza) sa različitim nivoima antioksidativne zaštite. Rezultati su ukazali na povećanu potrebu za antioksidativnom zaštitom kod pacijenata koji podležu hemodijalizi.

Uvod: Cilj istraživanja bio je ispitati razinu oksidativnog stresa korištenjem „AGE Reader“ uređaja i krutost krvnih žila korištenjem „Agedio B900“, te njihovu međusobnu povezanost u populaciji bolesnika s KBB. Sekundarni cilj bio je istražiti korelacije između AGE, PWv, CSAT, CDAT i svih mjernih parametara za skupinu bolesnika s KBB. Materijali i metode: Istaživanje je provedeno na 96 bolesnika s KBB, od kojih je 57 (59%) muškaraca i 39 (41%) žena. Medijan dobi svih bolesnika je 64 godine (interkvartilnog raspona od 51 do 72 godine) u rasponu od 20 do 84 godina. Glavni ispitivani parametri za svakog ispitanika s KBB bili su: opseg struka (cm), opseg bokova (cm), opseg nadlaktice (cm), dob i spol ispitanika, tjelesna tjelesna masa (kg) i tjelesna visina (cm). Vrijednosti arterijskih tlakova izmjerene su pomoću „Agedio B900“ uređaja koji mjeri centralni i periferni arterijski tlak, hemodinamske parametre te procjenjuje krutost krvnih žila. Analiza sastava tijela dobivena je pomoću uređaja „Tanita MC780 Multi Frequency segmentnog analizatora sastava“. Vrijednost krajnjih produkata glikacije (AGEs) u koži bolesnika mjerena uz pomoć „AGE Reader“ uređaja. Rezultati: U ovom istraživanju 69 pacijenata s KBB (73%) boluje od AH. Analizirajući razlike između muškaraca i žena nije opažena statistički značajna razlika u učestalosti, dobi prilikom dijagnoze i duljini liječenja AH u populaciji bolesnika s KBB. Među promatrani laboratorijskim parametrima, nađeno je kako je kod žena vrijednost HDL-a statistički značajno veća nego kod muškaraca (P, Introduction: The aim of the research was to examine the level of oxidative stress using the "AGE Reader" device and the stiffness of blood vessels using the "Agedio B900", and their correlation in the population of patients with CKD. The secondary objective was to investigate correlations between AGE, PWv, CSAT, CDAT and all measurement parameters for the group of patients with CKD. Materials and methods: The study was conducted on 96 patients with CKD, of which 57 (59%) were men and 39 (41%) were women. The median age of all patients is 64 years (interquartile range from 51 to 72 years), in the range from 20 to 84 years. The main investigated parameters for each subject with CKD were: waist circumference (cm), hip circumference (cm), upper arm circumference (cm), subject's age and sex, body mass (kg) and body height (cm). Arterial pressure values were measured using the "Agedio B900" device, which measures central and peripheral arterial pressure, hemodynamic parameters, and assesses the stiffness of blood vessels. Body composition analysis was obtained using the "Tanita MC780 Multi Frequency segmental composition analyzer" device. The value of glycation end products (AGEs) in the patient's skin measured with the help of the "AGE Reader" device. Results: In this study, 69 patients with CKD (73%) suffered from AH. Analyzing the differences between men and women, no statistically significant difference was observed in the frequency, age at diagnosis and length of treatment of AH in the population of patients with CKD. Among the observed laboratory parameters, it was found that the value of HDL was statistically significantly higher in women than in men (P

Nanotehnologija danas ima ulogu rastuće važnosti u razvoju novih industrijskih i medicinskih proizvoda. Nanočestice srebra (nanoAg) jedne su od najviše istraživanih tipova nanomaterijala za potencijalnu primjenu u medicini zbog svojih izraženih antimikrobnih svojstava. Njihova uporaba u medicinskom kontekstu ograničena je zbog štetnog utjecaja kojeg mogu imati na ljudske stanice i tkiva. Iako je toksičnost nanočestica srebra dobro poznata i istražena, manjkaju informacije o kombiniranim biološkim učincima nanoAg i drugih materijala ili nanomaterijala. Za razvoj novih, sigurnih uređaja i pomagala u kojima se koristi nanoAg, nužno je ispitati zdravstvene rizike koji mogu nastati međudjelovanjem nanoAg i materijala koji se primjenjuju u tim proizvodima. Polistiren je biološki inertan materijal široke industrijske i medicinske primjene, te su zbog toga nanočestice polistirena (nanoPS) odabrane za ispitivanje učinka mješavina nanočestica na modelne stanice ljudskog imunosnog sustava, Jurkat i THP-1. Nanočestice su karakterizirane metodama atomske mikroskopije sila te dinamičkog i elektroforetskog raspršenja svjetlosti. Analiziran je utjecaj nanoAg i nanoPS na preživljenje stanica, pokretanje apoptoze, parametre oksidativnog stresa, oštećenje DNA, pokretanje upalnog odgovora te promjene mehaničkih svojstava stanica. Unos nanočestica vizualiziran je konfokalnim mikroskopom i kvantificiran protočnim citometrom. Pokazano je da nanoAg i nanoPS ne stupaju u interakcije u vodenoj suspenziji ili u staničnom mediju, te da su mješavine nanoAg i nanoPS imale štetniji učinak na stanice Jurkat i THP-1 nego pojedinačne vrste nanomaterijala. Ovi rezultati važan su doprinos razumijevanju učinaka nanomaterijala na ljudsko zdravlje i razvoju novih i sigurnih primjena nanotehnologije u medicini.

Over the past decades plastics have become one of the largest contributors to marine pollution. Concerns have been increasing about ecotoxicological effects, not only from macro- and micro-scale plastics but also more recently from nanosized particles (1-100 nm). Moreover, understanding the consequences of nanoplastics release, particularly due to their high potential for bioaccumulation and biomagnification along marine food chains, is of growing importance. The work reported herein was focused towards the synthesis of polystyrene nanoparticles (PS NPs) and investigation of their impact on mussel Mytilus galloprovincialis. Mussels were exposed to PS NPs at concentrations of 0, 10, 50 and 100 mg L-1 for 96 h followed by a recovery period of 96 h. Oxidative stress markers, including enzymatic activity of catalase, glutathione S- transferase and acetylcholinesterase, and lipid peroxidation, were measured both after exposure and recovery period. The PS NPs have induced modulatory effects on the enzymes' activities while analysis of the same biomarkers after the recovery period showed values closer to controls. These data indicate that nanoplastics may give rise to deleterious effects in mussels after short-term exposure through generation of oxidative stress. However, the mussels also indicate an ability to reduce oxidative stress during the recovery period, likely due to activation of oxidative stress defence mechanisms and the gradual clearing of the nanoplastics by depuration.

U poslednje vreme, interesovanje za primenu biljnih ekstrakata u prehrambenoj i farmaceutskoj industriji nastavlja da raste, uglavnom zbog izraženih bioloških svojstava i efekta na zdravlje ljudi. Lekovite biljke i njihovi sekundarni metaboliti se progresivno koriste u lečenju raznih bolesti još od davnina. U ovom radu ispitivan je polifenolni profil biljnih metabolita i antioksidativna aktivnost 19 biljaka koje se tradicionalno koriste u lečenju upalnih procesa. Fenolna jedinjenja su ekstrahovana iz biljnog materijala ekstrakcijom potpomognutom ultrazvukom primenom dva konvencionalna organska rastvarača kao što su etanol i etil-acetat. Ispitivana je antioksidativna aktivnost dobijenih ekstrakata u cilju pronalaženja najaktivnijih biljnih ekstrakata, dok je visoko – efikasna tankoslojna hromatografija praćena analizom slike hromatograma primenjena u cilju pronalaženja sličnosti među biljnim vrstama. Optimalno razdvajanje fenolnih jedinjenja postignuto je primenom normalno-faznog sistema koga čini silica gel – etil-acetat/toluen/mravlja kiselina/voda (16/4/3/2), dok su za derivatizaciju korišćena tri reagensa (vanilin-sumporna, Neu-ov reagens i DPPH). Slike dobijenih hromatograma procesuirane su primenom programa ImageJ, dok su dalje numerički podaci analizirani multivarijantnom tehnikom.