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Predmet rada ove doktorske disertacije je razvoj analitičkih postupaka za praćenje relativnog sadržaja ukupnih proteina i ukupnih fenola u različitim uzorcima meda radi procene uticaja biotičkog stresa na pčelinja društva usled zaraze parazitima Varroa destructor i Nosema ceranae. Pored proizvodnje meda, pčele imaju veoma važnu ulogu u oprašivanju biljaka. Poslednjih godina u svetu dolazi do znatnog smanjenja pčelinjih društava, a jedan od razloga zbog kog dolazi do ove pojave su bolesti pčela. U literaturi je malo podataka o kvalitetu meda koji potiče iz pčelinjih zajednica izloženih biotičkom stresu. U cilju nalaženja indikatora u medu za procenu stepena zaraženosti pčelinjih društava, prikupljeni su uzorci meda iz košnica zaraženih varoom i nozemom, a zatim analizirani odabranim biohemijskim i fizičkohemijskim metodama. Određena je vrsta i stepen infekcije košnica pčelinjih društava iz kojih su prikupljeni uzorci meda. Polenskom analizom je određena botanička vrsta meda i nije utvrđena direktna veza između prisustva i koncentracije polena u uzorcima meda i stepena zaraženosti košnica. Uzorci meda su okarakterisani fizičkohemijskim metodama. Određeni su specifična optička rotacija, električna provodljivost, sadržaj vlage, slobodna kiselost i sadržaj pojedinačnih šećera. Profil šećera u uzorcima meda određen je visokoefikasnom jonskom hromatografijom sa elektrohemijskom detekcijom (HPAEC-PAD). Analiza glavnih komponenata (PCA) je upotreblјena za utvrđivanje razlika između medova koji potiču iz košnica različitog stepena zaraženosti na osnovu dobijenih fizičkohemijskih parametara. Rezultati analize glavnih komponenata su pokazali da sa porastom infekcije košnica u uzorcima meda raste sadržaj vode dok slobodna kiselost, apsolutna vrednost specifične optičke rotacije i električna provodljivost opadaju. Veći sadržaj fruktoze, melbioze i melezitoze izmeren je u uzorcima meda iz košnica višeg stepena infekcije u odnosu na uzorke meda koji potiču iz košnica manje zaraženosti. Sadržaj glukoze i izomaltoze bio je veći u uzorcima meda koji potiču od pčelinjih društava manje zaraženosti u odnosu na uzorke meda iz košnica veće zaraženosti. U cilju ispitivanja varijacije sadržaja biljnih i pčelinjih proteina u medu primenjene su elektroforetska analiza i određivanje aktivnosti enzima. Pomoću natrijum dodecilsulfat poliakrilamid gel elektroforeze (SDS PAGE) nije utvrđena značajna razlika u proteinskom profilu analiziranih uzoraka. Stoga je primenjena spektrofotometrijska metoda određivanja aktivnosti specifičnih enzima, katalaze i dijastaze, kao biljnog odnosno pčelinjeg marker enzima. Rezultati korelacione analize su pokazali da je stepen zaraze košnica u pozitivnoj korelaciji sa aktivnošću katalaze i može biti potencijalno koristan indikator za skrining meda poreklom iz košnica zaraženih varoom i nozemom. Pored toga, nije utvrđena promena u aktivnosti dijastaze u uzorcima meda čije je poreklo od društava koja su zaražena. Fluorescentna spektroskopija u kombinaciji sa naprednim statističkim metodama multivarijaciona rezolucija krivih – naizmenični najmanji kvadrati (MCR-ALS) i paralelna faktorska analiza (PARAFAC) je korišćena za određivanje relativnog sadržaja proteina i fenola u uzorcima meda... Protein and phenol content in honey samples as indicators of the influence of stress factors on honey bee colonies Abstract The subject of this doctoral dissertation is the development of analytical procedures for monitoring the relative content of total proteins and total phenols in different honey samples to assess the impact of biotic stress on honey bee colonies due infected with parasites Varroa destructor and Nosema ceranae. In addition to honey production, bees have very important role in pollinating plants. In recent years, there has been a significant decrease in bee colonies in the world and one of the reasons why this phenomenon occurs is bee diseases. In the literature, the data are not abundant on the honey quality change related to bee colonies exposure to biotic stress. In order to find markers in honey to assess the degree of infection of bee colonies, honey samples were collected from hives infected with parasites V. destructor and N. ceranae parasites and analyzed by selected biochemical and physicochemical methods. The type and degree of infection of bee colonies related to the analyzed honey samples were determined. Pollen analysis has proved the botanical type of honey samples and it was observed that the presence and concentration of pollen in honey samples were not correlated with the infestation of hives. Honey samples were characterized by physicochemical methods. Specific optical rotation, electrical conductivity, moisture content, free acidity, and sugar content were determined to obtain additional characteristics of honey samples. Sugar profile in honey samples was determined by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Principal component analysis (PCA) was used to determine differences between honey samples originating from the hives with different infection levels. The obtained results showed that in hives with the increase of infection, in honey samples the water content increased, while the free acidity, the absolute value of the specific optical rotation, and the electrical conductivity decreased. The higher content of fructose, melbiosis and melezitose was observed in honey samples from the hives with a higher degree of infection in comparison with honey samples from the hives with the lower infection degree. Glucose and isomaltose content was higher in honey samples originating from the bee colonies less infected compared to honey samples from the hives with the higher infection. In order to investigate variations in the content of plant and bee protein markers in honey, biochemical methods such as electrophoretic analysis and determination of enzyme activity were applied. Using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS PAGE), no significant difference was found in the protein profile of the analyzed honey samples. Therefore, the spectrophotometric method was applied for determination of the activity of specific enzymes, catalase and diastase, as plant and bee marker enzymes, respectively. The results of correlation analysis showed that the colonies’ infestation level was positively correlated with the activity of catalase which can be a useful indicator for screening of honey originating from beehives infested by V. destructor and N. ceranae. On the other hand, no change in diastase activity was found in honey samples obtained from infected societies. Fluorescence spectroscopy in combination with advanced statistical methods Multivariate Curve Resolution - Alternating Least Squares (MCR-ALS) and Parallel Factor Analysis (PARAFAC) was used to determine the relative protein and phenol content in honey samples...

U ovoj doktorskoj disertaciji je ispitivano vezivanje tigeciklina za humani serumski albumin i interakcije tigeciklina sa specifičnim markerima vezujućih mesta (varfarinom i ibuprofenom), jonima metala (Ca2+, Cu2+ i Fe3+) i fluorohinolonima (sparfloksacinom, levofloksacinom i ciprofloksacinom) koje se javljaju na nivou vezivanja za humani serumski albumin. Sve interakcije su ispitivane u simuliranim fiziološkim uslovima in vitro. Korišćene su spektroskopske metode (fluorescentna i apsorpciona spektroskopija) i AutoDock analiza. Gašenje fluorescencije aktivnog centra humanog serumskog albumina (HSA) tigeciklinom (TGC) je posledica formiranja HSA-TGC kompleksa, što ukazuje da je mehanizam gašenja fluorescencije statički proces. Konstante vezivanja (Ka) HSATGC sistema su bile 1,8 × 104; 4,0 × 105 i 4,5 × 106 L/mol na 298, 303 i 308 K, a broj vezujućih mesta (n) je izračunat na oko 1, što ukazuje da HSA i TGC formiraju komplekse u molskom odnosu približno 1:1. Prema van’t Hofovoj jednačini, vrednosti promene entalpije (ΔH0) i promene entropije (ΔS0) su bile 423,2 kJ/mol i 1502,3 J/mol·K, što ukazuje da tipične hidrofobne interakcije igraju najvažniju ulogu u reakciji vezivanja i stabilizaciji HSA-TGC kompleksa. Rastojanje vezivanja (r) između donora (HSA) i akceptora (TGC) je izračunato da iznosi 3,63 nm prema Forsterovoj teoriji rezonantnog energetskog transfera. Glavno vezujuće mesto za tigeciklin je mesto I smešteno u hidrofobnoj šupljini subdomena IIA. Mesto I je imalo prednost nad mestom II. Joni metala (Ca2+, Cu2+ i Fe3+) su indukovali povećanje afiniteta tigeciklina za humani serumski albumin. Tigeciklin je doveo do smanjenja afiniteta fluorohinolona (SPF, LVF i CPF) za humani serumski albumin. Ova doktorska disertacija treba da pruži bolje razumevanje vezivanja tigeciklina za humani serumski albumin i poboljša njegovu primenu u kliničkoj praksi. Rezultati ove doktorske disertcije mogu biti od velikog značaja za dalji razvoj prirodnih nauka, kliničke farmakologije i medicine. In this doctoral thesis the binding of tigecycline for human serum albumin and interactions of tigecycline with specific markers of binding sites (warfarin and ibuprofen), metal ions (Ca2+, Cu2+ and Fe3+) and fluoroquinolones (sparfoxacin, levofloxacin and ciprofloxacin) which occur at the level of binding for human serum albumin were investigated. All interactions were studied in simulated physiological conditions in vitro. The spectroscopic methods (fluorescence and absorption spectroscopy) and AutoDock analysis were used. Fluorescence quenching of the active center of human serum albumin (HSA) by tigecycline (TGC) is due to the formation of HSA-TGC complex, indicating that the mechanism of fluorescence quenching is a static process. The binding constants (Kа) of HSA-TGC system were 1.8 × 104, 4.0 × 105 and 4.5 × 106 L/mol at 298, 303 and 308 K, and the number of binding sites (n) was calculated at about 1, indicating that HSA and TGC formed complexes in a molar ratio of approximately 1:1. According to the van't Hof equation, the values of the enthalpy change (ΔH0) and the entropy change (ΔS0) were 423.2 kJ/mol and 1502.3 J/mol·K, indicating that typical hydrophobic interactions play a major role in the binding reaction and stabilization of HSA-TGC complex. The binding distance (r) between the donor (HSA) and the acceptor (TGC) was calculated to be 3.63 nm according to Förster resonance energy transfer theory. The major binding site for tigecycline is the site I located in the hydrophobic cavity of subdomain IIA. Тhe site I had an advantage over site II. Metal ions (Ca2+, Cu2+ and Fe3+) induced an increase in the affinity of tigecycline for human serum albumin. Tigecycline has led to a decrease in the affinity of fluoroquinolones (SPF, LVF and CPF) for human serum albumin. This doctoral thesis should provide a better understanding of the binding of tigecycline for human serum albumin and improve its application in clinical practice. The results of this doctoral thesis can be of great importance for the further development of natural sciences, clinical pharmacology and medicine.

U ovoj doktorskoj disertaciji ispitan je uticaj antitumorskih mononuklearnih i binuklearnih kompleksa zlata(III) (AubipyC, Aubipy(OH)2, Aupy(OAc)2, Auoxo6, Au2bipyC i Au2phen) na Na+/K+-ATPazu, kao i uticaj polioksometalata, 12- volframsilicijumove i 12-volframfosforne kiseline (WSiA i WPA) na acetilholinesterazu (AChE). Primenom različitih fizičkohemijskih metoda (UV/Vis i fluorescentna spektroskopija, cirkularni dihroizam, elektronska paramagnetna rezonantna (EPR) spektroskopija) u kombinaciji sa molekulskim modelovanjem, dobijen je detaljan uvid u njihovu interakciju na molekulskom nivou. Ispitivanjem uticaja ovih jedinjenja na antioksidativne enzime i na nivo malondialdehida, procenjeni su njihovi toksični potencijali, a merenjem učestalosti mikronukleusa i indeksa ćelijske proliferacije procenjeni su njihovi genotoksični i citotoksični efekti... In this doctoral dissertation, the interaction of antitumor mononuclear and binuclear gold(III) complexes (AubipyC, Aubipy(OH)2, Aupy(OAc)2, Auoxo6, Au2bipyC and Au2phen) with Na+/K+-ATPas,e as well as the interaction of polioxotungstates, 12- tungstosilicic acid and 12-tungstophosphoric acid (WSiA and WPA), with acetylholinesterase have been investigated. Various spectroscopic methods (UV/Vis and fluorescent spectroscopy, circular dichroism, electron paramagnetic resonance (EPR) spectroscopy) in combination with docking studies were used in order to obtain detailed insight into their interaction on the molecular level. By investigating the influence of these compounds on the antioxidative enzymes and the level of malondialdehyde, their toxic potentials were evaluated. Furthermore, their genotoxic and cytotoxic effects were estimated by measuring the incidence of micronuclei and the cytokinesis-block proliferation index in human lymphocyte cultures...

U ovoj tezi su primenjene hemometrijske metode u dekompoziciji fluorescentnih i FTIR spektara, dobijenih snimanjem uzoraka nanočestica, proteina i bioloških uzoraka. U cilju dekompozicije spektralnih podataka korišćene su: multivarijaciona rezolucija krivih, analiza glavnih komponenti i paralelna faktorska analiza. Ove analize su rađene u cilju: (i) karakterisanja nanočestica (kvantnih i karbonskih tačaka) koje imaju različite vrste primena, (ii) proučavanja strukturnih promena ćelijskog zida koje nastaju usled mutacija u biosintezi njegovih konstitutivnih polimera u cad-c cad-d mutantu u odnosu na divlji tip Arabidopsis thaliana, (iii) praćenja strukturnih prelaza kod proteina goveđi serum albumin (BSA) usled interakcije sa ATP-om. Multivarijaciona rezolucija krivih je pokazala da se struktura nanosenzora ZnS:Mn2+ ne menja funkcionalizacijom sa dendrimerom PAMAM-OH. Ovo ukazuje da se ovaj nanosenzor može koristiti za detekciju i kvantifikaciju Cd2+. Ista hemometrijska metoda je pokazala da je kompleks CDs-MSA stabilan nakon dodavanja Ag+ jona, što ukazuje na veliku selektivnost ovog senzora u detekciji Ag+ jona... In this thesis chemometric methods in decomposition of fluorescent and FTIR spectra were applied. The spectra were obtained by recording samples of nanoparticles, proteins and biological samples. Decomposition of spectral data was performed by using: multivariate curve resolution, principal components analysis and parallel factor analysis. These analyses were carried out in order to: (i) characterize nanoparticles (quantum and carbon dots) that have a variety of applications, (ii) study the structural changes in the cell wall due to mutations in the biosynthesis of its constituent polymers in cad-c cad-d mutant of Arabidopsis thaliana and compare with the wild-type, (iii) monitor the structural transition in the case of the protein bovine serum albumin (BSA) as a result of interaction with ATP. Multivariate curve resolution showed that the structure of nanosensors ZnS:Mn2+ does not change by funkcionalization with PAMAM-OH dendrimer. This indicates that this nanosensor can be used for detection and quantitation of Cd2+. The same chemometric methods showed that the complex MSA-CDs is stable after addition of Ag+ ions, which indicates a high selectivity of the sensor in detection of Ag+ ions. In the study of fluorescent CdSe quantum dots doped with phosphate porous heterostructures (PPH-NH2@CdSe), multivariate curve resolution showed that the CdSe emission band does not change under the influence of the solid support (that are photostable) and can be used as an agent for labeling finger prints. This photostability of PPH-NH2@CdSe proved to be a feature that improves image quality of latent fingerprints on non-porous surfaces, compared to the usual forensic techniques...

Kombinacija optičkih spektroskopskih tehnika i odgovarajućih metoda za analizu i modeliranje spektralnih podataka se pokazala kao veoma obećavajuća tehnika za karakterizaciju i analizu organskih i neorganskih kompleksnih sistema. Razumevanje fizičkih fenomena koji se javljaju u kompleksnim sistemima predstavlja bitan preduslov za primenu spektroskopskih tehnika u različitim oblastima nauke. U ovoj tezi su izmerene fluorescentne karakteristike četiri kompleksna sistema: tri organska (rastvor vode i aminokiselina, med i tkivo dojke) i jedan neorganski (kompleksni sistem fosfora na bazi retkih zemalja). Dobijeni fluorescentni podaci su modelirani primenom paralelne faktorske analize. Izgrađeni modeli su omogućili definisanje fluorescirajućih komponenti (fluorofora) prisutnih u sistemima, pri čemu je model kao rezultat dao čiste spektre svake pojedinačne fluorofore, kao i njihove relativne koncentracije u svakom od uzoraka sistema. U neorganskom sistemu i rastvoru aminokiselina broj fluorofora i njihove koncentracije su unapred bile poznate i na taj način je testirana uspešnost modela kao i njegova sposobnost predikcije prisustva i koncentracija fluorofora u nepoznatim uzorcima. Pokazano je da je dobijeni model veoma uspešan, sa odstupanjem od samo 0.036% od stvarne koncentracije kod neorganskog sistema. Za sistem meda, model je pokazao da je u sistemu prisutno 6 fluorofora i dobijeni su čisti spektri svake od njih kao i njihove relativne koncentracije u svakom pojedinačnom uzorku. Dalje analize spektara meda su ukazale da je na osnovu koncentracija fluorofora moguće odrediti botaničko poreklo meda. Spektri meda su zatim analizirani metodom glavnih komponenata kao i diskriminantnom analizom parcijalno najmanjih kvadrata. Rezultati su pokazali veliku uspešnost primene metode fluorescentne spektroskopije za klasifikaciju i autentifikaciju uzoraka meda. Proračunom modela tkiva dojke je ustanovljeno da postoje 4 dominantne fluorofore. Utvrđeno je da je na osnovu njihovih koncentracija moguće sa tačnošću od 100% odrediti vrstu promena na tkivu (benigno ili maligno). Rezultati su potom poređeni sa rezultatima analize potpornih vektora primenjenih na neobrađene spektre, na osnovu kojih je pokazano da se sa istim podacima ne može dobiti podjednako uspešan model... The combination of optical spectroscopic techniques and appropriate methods for analysis and modeling of spectral data proved to be a very promising technique for the characterization and analysis of organic and inorganic complex systems. Understanding of physical phenomena that occur in complex systems is an essential prerequisite for the application of spectroscopic techniques in various fields of science. Fluorescence characteristics of four complex systems: three organic (solution of water and amino acids, honey and breast tissue) and one inorganic (a complex system of phosphorus-based rare earth) were measured in this thesis. The obtained fluorescence data were modeled using parallel factor analysis. The constructed models have enabled definition of the fluorescing components (fluorophores) present in the system, wherein the model as a result gave pure spectra of the individual fluorophore as well as their relative concentrations in each sample of the system. In the inorganic system and solution of amino acids, the number of fluorophores and their concentrations were known in advance, and thus the performance of the model was tested as well as its ability to predict the presence and concentrations of fluorophores in the unknown samples. It was shown that the resulting model is very successful, with a deviation of only 0.036% of the actual concentration in inorganic systems. For the system of honey, the model showed that 6 fluorophores are present in the system and their pure spectra were obtained, as well as their relative concentrations in each sample. Further analysis of the spectra of honey have demonstrated that based on the concentrations of the fluorophores the botanical origin of honey can be determined. Spectra of honey were then analyzed using principal component analysis and discriminant partial least squares. Results have shown great success in application of fluorescence spectroscopy methods for classification and authentication of honey samples. Based on calculated model of the breast tissue it was determined that there are four dominant fluorophores. It was estabilshed that based on their concentrations it possible to determine the type of change in the tissue (benign or malignant) with an accuracy of 100%. The results were then compared with the results of support vector machine analysis applied to the raw spectra, from which it was shown that using the same data successful model can not be obtained (classification error 35.71%). However, it has been shown that with...

Kombinacija optičkih spektroskopskih tehnika i odgovarajućih metoda za analizu imodeliranje spektralnih podataka se pokazala kao veoma obećavajuća tehnika zakarakterizaciju i analizu organskih i neorganskih kompleksnih sistema. Razumevanjefizičkih fenomena koji se javljaju u kompleksnim sistemima predstavlja bitan preduslovza primenu spektroskopskih tehnika u različitim oblastima nauke. U ovoj tezi su izmerenefluorescentne karakteristike četiri kompleksna sistema: tri organska (rastvor vode iaminokiselina, med i tkivo dojke) i jedan neorganski (kompleksni sistem fosfora na baziretkih zemalja). Dobijeni fluorescentni podaci su modelirani primenom paralelnefaktorske analize. Izgrađeni modeli su omogućili definisanje fluorescirajućih komponenti(fluorofora) prisutnih u sistemima, pri čemu je model kao rezultat dao čiste spektre svakepojedinačne fluorofore, kao i njihove relativne koncentracije u svakom od uzorakasistema. U neorganskom sistemu i rastvoru aminokiselina broj fluorofora i njihovekoncentracije su unapred bile poznate i na taj način je testirana uspešnost modela kao injegova sposobnost predikcije prisustva i koncentracija fluorofora u nepoznatimuzorcima. Pokazano je da je dobijeni model veoma uspešan, sa odstupanjem od samo0.036% od stvarne koncentracije kod neorganskog sistema. Za sistem meda, model jepokazao da je u sistemu prisutno 6 fluorofora i dobijeni su čisti spektri svake od njih kaoi njihove relativne koncentracije u svakom pojedinačnom uzorku. Dalje analize spektarameda su ukazale da je na osnovu koncentracija fluorofora moguće odrediti botaničkoporeklo meda. Spektri meda su zatim analizirani metodom glavnih komponenata kao idiskriminantnom analizom parcijalno najmanjih kvadrata. Rezultati su pokazali velikuuspešnost primene metode fluorescentne spektroskopije za klasifikaciju i autentifikacijuuzoraka meda. Proračunom modela tkiva dojke je ustanovljeno da postoje 4 dominantnefluorofore. Utvrđeno je da je na osnovu njihovih koncentracija moguće sa tačnošću od100% odrediti vrstu promena na tkivu (benigno ili maligno). Rezultati su potom poređenisa rezultatima analize potpornih vektora primenjenih na neobrađene spektre, na osnovukojih je pokazano da se sa istim podacima ne može dobiti podjednako uspešan model... The combination of optical spectroscopic techniques and appropriate methods foranalysis and modeling of spectral data proved to be a very promising technique for thecharacterization and analysis of organic and inorganic complex systems. Understandingof physical phenomena that occur in complex systems is an essential prerequisite for theapplication of spectroscopic techniques in various fields of science. Fluorescencecharacteristics of four complex systems: three organic (solution of water and amino acids,honey and breast tissue) and one inorganic (a complex system of phosphorus-based rareearth) were measured in this thesis. The obtained fluorescence data were modeled usingparallel factor analysis. The constructed models have enabled definition of the fluorescingcomponents (fluorophores) present in the system, wherein the model as a result gave purespectra of the individual fluorophore as well as their relative concentrations in eachsample of the system. In the inorganic system and solution of amino acids, the number offluorophores and their concentrations were known in advance, and thus the performanceof the model was tested as well as its ability to predict the presence and concentrations offluorophores in the unknown samples. It was shown that the resulting model is verysuccessful, with a deviation of only 0.036% of the actual concentration in inorganicsystems. For the system of honey, the model showed that 6 fluorophores are present inthe system and their pure spectra were obtained, as well as their relative concentrationsin each sample. Further analysis of the spectra of honey have demonstrated that based onthe concentrations of the fluorophores the botanical origin of honey can be determined.Spectra of honey were then analyzed using principal component analysis and discriminantpartial least squares. Results have shown great success in application of fluorescencespectroscopy methods for classification and authentication of honey samples. Based oncalculated model of the breast tissue it was determined that there are four dominantfluorophores. It was estabilshed that based on their concentrations it possible to determinethe type of change in the tissue (benign or malignant) with an accuracy of 100%. Theresults were then compared with the results of support vector machine analysis applied tothe raw spectra, from which it was shown that using the same data successful model cannot be obtained (classification error 35.71%). However, it has been shown that with...