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Biljna vrsta Euphorbia palustris pripada rodu Euphorbia iz familije Euphorbiaceae. Ove biljke koristе se od davnina u tradicionalnoj medicini mnogih zemalja sveta. Iz lateksa različitih vrsta roda Euphorbia izolovani su brojni terpenski derivati koji imaju širok spektar bioloških aktivnosti, od kojih su neke veoma značajne zbog potencijalne mogućnosti primene u savremenoj medicini i dizajnu lekova. Ispitivanja različitih derivata triterpena pokazala su da, pored nekih neželjenih efekata kao što je zapaljenje kože, određeni derivati mogu imati antikancerogeno i anti-HIV dejstvo. Cilj ovog istraživanja bio je ispitivanje lateksa biljne vrste Euphorbia palustris iz kojeg su izolovana, prečišćena i okarakterisana dva triterpenska derivata: (+)-tirukalol i (+)-obtusifoliol.

Cilj ovog master rada je razvoj objedinjene sinteze dva tipa tricikličnih terpena: cedrena i silfinena, preko zajedničkog bicikličniog intermedijera dobijenog intramolekulskom aldolnom kondenzacijom. Sintetički put divergira kod reakcije alkilovanja. Proizvod alkilovanja sa 4-brom-1-butenom nije dobijen. Alkilovanje sa strukturnim homolozima, alil-bromidom i 5-brom-1-pentenom, je dalo odgovarajuće proizvode. Iz alilovanog proizvoda, dobijen je silfinenski intermedijer.

Biljna vrsta Euphorbia palustris pripada rodu Euphorbia iz familije Euphorbiaceae. Ove biljke koristile su se u tradicionalnoj medicini različitih zemalja sveta. Iz lateksa različitih vrsta roda Euphorbia izolovani su mnogi diterpeni i triterpeni koji imaju širok spektar bioloških aktivnosti, od kojih su neke veoma značajne zbog potencijalne mogućnosti primene u savremenoj medicini. Ispitivanja derivata tiglijanskih diterpena pokazala su da pored nekih neželjenih efekata kao što je zapaljenje kože, određeni derivati mogu imati antikancerogeno i anti-HIV dejstvo. Cilj rada bio je ispitivanje lateksa biljne vrste Euphorbia palustris iz kojeg su izolovani, prečišćeni i okarakterisani jedan tiglijanski diterpen i jedan cikloartanski triterpen.

Vrsta Candida albicans jedan je od najčešćih uzročnika gljivičnih infekcija ljudi. U okviru ove doktorske disertacije ispitivani su mehanizmi antifungalnog delovanja 9 flavonoida (apigenin, apigetrin, astragalin, kvercetin, kvercitrin, izokvercitrin, rutin, luteolin i viteksin), 2 terpena (kamfor i eukaliptol) i 4 nitratna estra heterocikličnih jedinjenja (N-(2-nitrooksietil)-1Η-indol-2-karboksamid (MK55), Ν-(2-nitroksietil)-hinolin-2-karboksamid (MK56), Ν-(2-nitroksietil)-6-(piperidin-1-il)-9H-purin acetamid (MK94), 5΄-Ο-nitro adenozin (MK129)). Najbolju antifungalnu aktivnost ispoljili su nitratni estri heterocikličnih jedinjenja. U smanjenju razvoja virulentnosti ove gljive, nastanku biofilma i formiranju hifalnih ćelija, istakla se aktivnost flavonoida, posebno apigetrina, apigenina i izokvercitrina. Razvoj antifungalne rezistencije C. albicans povezan je sa povećanjem aktivnosti efluks pumpi kodiranih genima MDR1, CDR1 i CDR2. Promene u ekspresiji ovih gena zabeležene su nakon tretmana astragalinom i apigeninom (smanjenje ekspresije CDR1), kamforom (povećanje ekspresije CDR1, smanjenje ekspresije CDR2) i eukaliptolom (povećanje ekspresije CDR1 i CDR2), ukazujući na potencijalna korisna svojstva flavonoida, ali i moguće neželjene posledice primene testiranih terpena. Rezistencija je povezana i sa povećanom ekspresijom ERG11, gena koji kodira protein lanosterol 14α-demetilazu (CYP51), ciljno mesto azolnih lekova. Izokvercitrin i MK94 izazivaju neželjeno povećanje, dok MK55 redukuje nivo ekspresije ovog gena. Mehanizam antifungalnog delovanja MK55 obuhvata i inhibiciju fungalnog proteina CYP51. Narušavanje integriteta ćelijske membrane, kao potencijalnog mehanizma delovanja, potvrđeno je za apigenin, apigetrin i astragalin. Upotreba MK55, MK94, apigenina, apigetrina, rutina, kvercetina, kvercitrina, izokvercitrina, luteolina i kamfora može se smatrati bezbednom, dok se eukaliptol zbog svog citotoksičnog efekta prema ćelijama jetre ne preporučuje za potencijalnu primenu u antifungalnoj terapiji. Candida albicans is among the most common human fungal pathogens. Mode of antifungal action has been tested for 9 flavonoids (apigenin, apigetrin, astragalin, quercetin, quercitrin, isoquercitrin, rutin, luteolin and vitexin), 2 terpens (camphor and eucalyptol) and 4 nitrate esters of heterocyclic compounds (N-(2-nitooxyethyl)-1Η-indol-2-carboxamide (MK55), Ν-(2-nitrooxyethyl)-quinoline-2-carboxamide (MK56), Ν-(2-nitrooxyethyl)-6-(piperidin-1-yl)-9H-purine acetamide (MK94), 5΄-Ο-nitroadenosine (MK129)). The most prominent anticandidal activity has been proven for nitrate esters of heterocyclic compounds. Significant activity of flavonoids, especially apigetrin, apigenin and isoquercitrin, could be noticed in reducing fungal virulence, biofilm formation and hyphal growth. Resistance to antifungal therapy is linked to increased activity of C. albicans efflux pumps encoded by MDR1, CDR1 and CDR2. Disturbance in expression levels of these genes has been observed after treatment with astragalin and apigenin (decreased CDR1 expression), camphor (increased CDR1 and decreased CDR2 expression) and eucalyptol (increased CDR1 and CDR2 expression levels), suggesting possible beneficial antimicrobial effects of flavonoids as well as potential harmful consequences of selected terpenes application. Antifungal resistance is also linked to increased expression of ERG11, gene encoding for lanosterol 14α-demethylase (CYP51), target protein for azole antifungals. Treatment with isoquercitrin and MK94 led to increased expression of this gene, while treatment with MK55 reduced it. Mode of action for MK55 involves inhibition of CYP51 enzyme. Damage of fungal cell membrane as a potential mechanism of antifungal activity has been observed for apigenin, apigetrin and astragalin. Application of MK55, MK94, apigenin, apigetrin, rutin, quercetin, quercitrin, isoquercitrin, luteolin and camphor could be considered safe, based on the results of their cytotoxicity towards the liver cell line, while eucalyptol should be used with serious precaution.

In order to determine the terpenes effect on enzyme activity is been studied the impact of aqueous (prepared manually (I1) or ultrasound (I2) mixing) and DMSO (dimethyl sulfoxide) terpenes solution (carvacrol, linalool, nerol and geraniol) on the activity of creatine kinase, lactate dehydrogenase, alkaline phosphatase, gamma-glutamyl transferase, aspartate aminotransferase and alanine aminotransferase,as well as the impact of the same type of terpenes solution (carvacrol, linalool, nerol, geraniol, β-pinene, myrcene, citral, eucalyptol, terpineol, and eugenol) on the activity of amylase in the control sera (with the normal (N) and increased (H) activity of the ingredients. Measurement of activity was conducted after incubation of the serum sample from the 10 minutes and aqueous solutions of terpenes prepared as I1 or as I2, or solutions of terpenes in DMSO at four concentrations. All the terpenes solutions have demonstrated that the inhibitory effect depends on various parameters: kinds of serum (higher percentage of inhibition in N than the H serum), the type of solvent ( the aqueous solution as I2 and DMSO solutions showed approximately the same percentage of inhibition in both types of serum) and way of preparing the aqueous solutions (a higher percentage of inhibition have the aqueous solution of terpenes as I2 in N serum and as I1 in H serum), as well as on the type of terpenes or enzymes. Statistical data processing all terpenes solution (with the partial exception of neroli and geraniol) confirmed the grouping or by type of serum or by type of solvent or the method of preparation of aqueous solution.

U ovom radu analizirana je fitohemijska i molekularna varijabilnost i diferencijacija prirodnih populacija taksona sekcije Juniperus roda Juniperus (Cupressaceae) sa Balkanskog poluostrva... This thesis deals with phytochemical & molecular variability variability & differentation of natural population of taxa belonging to section Juniperus of the genus Juniperus (Cupressaceae) from the Balkan peninsula...

U ovom radu analizirana je fitohemijska i genetiţka varijabilnost i diferencijacija sedam autohtonih populacija Pinus nigra u Srbiji koje pripadaju razliţitim infraspecijskim taksonima (ssp. nigra, var. gocensis, ssp. pallasiana i var. banatica). U dvogodišnjim ţetinama 195 jedinki iz sedam populacija odreŤen je sadrţaj i varijabilnost n-alkana i terpena. Kutikularne voskove ţetina istraţivanih populacija crnog bora odlikuje serija nalkana od C16 do C33, osim u populacijama opisanim kao ssp. nigra u kojima je detektovana serija od C18 do C33. Najzastupljeniji su n-alkani sa neparnim brojem C atoma: C23, C25, C27 i C29 (proseţni sadrţaj 10.3, 16.5, 15.1 i 10.7%, respektivno). U etarskim uljima ţetina utvrŤeno je ukupno 75 komponenti, od koji je 58 identifikovano. Dominantne komponente su α-pinen (43.6%) i germakren D (29.8%), koje zajedno ţine 73.4% mase ulja. Multivariacione statistiţke analize (PCA, CDA i HCA) koriššenih fitohemijskih markera su, generalno, pokazale tendenciju formiranja tri grupe populacija (nigra, pallasiana i banatica). Na osnovu varijabilnosti terpena, populacije Priboj i Goţ (determinisane kao var. gocensis) pokazale su umerenu tendenciju odvajanja od prve grupe (ssp. nigra), dok je prema sastavu n-alkana populacija Priboj pokazala još vešu tendenciju odvajanja. Pretpostavljeno je da takson gocensis predstavlja tranzicionu formu izmeŤu taksona nigra na zapadu i pallasiana na jugoistoku Srbije. U cilju odreŤivanja genetiţkog diverziteta i diferencijacije sedam istraţivanih populacija P. nigra iz Srbije, testirano je 114 jedinki pomošu jedarnih (nuDNK), hloroplastnih (cpDNK) i mitohondrijalnih (mtDNK) markera koji imaju bi-parentalno, paternalno i maternalno nasleŤivanje, respektivno, kao i razliţitu brzinu evolucije. Zbog nedostatka jedarnih mikrosatelita (nuSSRs) specijalno razvijenih za P. nigra, uraŤena je inter-specijska PCR amplifikacija tri EST-SSRs razvijenih za P. taeda. Iako su dva ESTSSR lokusa uspešno PCR-amplifikovana u našim uzorcima, utvrŤeno je odsustvo varijabilnosti zbog ţega su iskljuţeni iz dalje analize. Od pet testiranih cpDNK SSRs, tri su uspešno PCR-amplifikovana u našim uzorcima, sa 22 detektovana alela koji formiraju 38 haplotipova. Kao što je i oţekivano, s obzirom na intenzivan protok gena posredstvom polena kod ţetinara, utvrŤen je veoma visok nivo cpDNK diverziteta (HT = 0.949) i mali stepen diferencijacije (Fst = 0.017, p = 0.108, Gst = 0.024, Rst = 0.066)... In this work phytochemical and genetic variability and differentiation of seven native populations belonging to different infraspecific taxa of Pinus nigra (ssp. nigra, var. gocensis, ssp. pallasiana i var. banatica) in Serbia were analyzed. In the needles of 195 trees from seven populations, composition and variability of n-alkanes and terpenes were determined. The size of the n-alkanes found in the needle cuticular waxes of the black pine populations investigated ranged from C16 to C33, with the exception of that of the subspecies nigra, which ranged from C18 to C33. The most abundant n-alkanes were the four odd-numbered n-alkanes C23, C25, C27, and C29 (mean contents of 10.3, 16.5, 15.1, and 10.7%, respectively). In the needle essential oils a total of 58 individual components out of the 75 compounds detected could be identified. The major components of the oils were α-pinene (43.6%) i germakrene D (29.8%), comprising together in average 73.4% of the essential oil composition. Multivariate statistical analysis (PCA, CDA, and HCA) of used phytochemical markers showed, in general, a tendency to the formation of three populations groups (nigra, pallasiana, and banatica). Based on the terpene variability, populations Priboj and Goţ, determined as var. gocensis, showed a moderate tendency of separation from the first group (ssp. nigra), while based on n-alkane composition, population Priboj showed even a greater tendency of separation. It was assumed that taxon gocensis might be a transitional form between taxa nigra occurring in the west and pallasiana growing in the southeast of Serbia. We screened 114 trees from seven studied P. nigra populations from Serbia with nuclear (nrDNA), chloroplast (cpDNA) and mitochondrial (mtDNA) markers which have bi-parental, paternal and maternal inheritance, respectively, and different evolutionary rates to infer levels of genetic diversity and structuring within studied area. Due to the lack of nuclear microsatellites (nuSSRs) specifically developed for P. nigra, we employed three EST-SSRs developed for P. taeda for cross-species amplification in P. nigra. Although two EST-SSR loci were successfuly PCR-amplified in our samples, they were invariable and discarded from further analyses...

In studies of population variability, particular attention has to be paid to the selection of a representative sample. The aim of this study was to assess the size of the new representative sample on the basis of the variability of chemical content of the initial sample on the example of a whitebark pine population. Statistical analysis included the content of 19 characteristics (terpene hydrocarbons and their derivates) of the initial sample of 10 elements (trees). It was determined that the new sample should contain 20 trees so that the mean value calculated from it represents a basic set with a probability higher than 95 %. Determination of the lower limit of the representative sample size that guarantees a satisfactory reliability of generalization proved to be very important in order to achieve cost efficiency of the research. Cilj ovih istraživanja je da se na osnovu varijabilnosti hemijskih svojstava početnog uzorka proceni veličina novog reprezentativnog uzorka jedne populacije munike. Statističkom analizom je obuhvaćen sadržaj 19 svojstava (terpenskih ugljovodonika i njihovih derivata) početnog uzorka od 10 elemenata (stabala). Utvrđeno je da novi uzorak treba da sadrži 20 stabala da bi srednja vrednost izračunata iz njega reprezentovala osnovni skup sa verovatnoćom većom od 95 %. Određivanje donje granice veličine reprezentativnog uzorka koja garantuje zadovoljavajuću pouzdanost uopštavanja je veoma važno zbog ekonomičnosti istraživanja. Projekat ministarstva br. OI-173011, br. TR-37002 i br. III-43007