Ecophysiological adaptations of Pinus nigra Arn., Аesculus hippocastanum L. and Platanus acerifolia (Ait.) Willd. species in urban environments with different sources of pollution

Urbana staništa su pod snažnim antropogenim uticajem i karakteriše ih intenzivno zagađenje vazduha, vode i zemljišta materijama poreklom iz različitih stacionarnih i mobilnih izvora. Specifična vrsta polutanata u ovim sredinama su metali koji se zbog svoje toksičnosti i biološke nerazgradivosti akumuliraju u zemljištu. Istraživanja u okviru ove disertacije su obavljena u urbanim parkovima u Pančevu, Smederevu, Obrenovcu i Beogradu koji su izloženi zagađivanju hemijskim elementima uključujući toksične metale poreklom iz različitih izvora. Obuhvatala su analize biljaka, kao i zemljišta na kojima rastu. Za ekofiziološka istraživanja odabrane su tri drvenaste vrste biljaka koje se dugi niz godina uspešno gaje na urbanim staništima: Pinus nigra Arn., Aesculus hippocastanum L. i Platanus acerifolia (Ait.) Willd. Osnovni cilj doktorske disertacije je bio definisanje faktora limitirajućih za razvoj biljaka, kao i ispitivanje njihovih ekofizioloških karakteristika i adaptivnog odgovora. Posebna pažnja je posvećena problemu zagađivanja metalima i metaloidima, njihovoj mobilnosti i dostupnosti biljkama, kao i njihovom uticaju na fiziološke procese u bilјkama (efikasnost fotosinteze i sadržaj fotosintetičkih pigmenata). Ispitivanja fizičko-hemijskih karakteristika zemljišta su pokazala da urbana zemljišta karakteriše visok udeo frakcije peska, nizak sadržaj higroskopne vlage, organskog ugljenika i azota, alkalna reakcija, ali i povoljan odnos C/N za mineralizaciju organske materije i oslobađanje minerala esencijalnih za biljke. Navedeni limitirajući faktori (toksičan sadržaj B, Cr, Ni i Pb izmerenih u zemljištu urbanih parkova) u sinergističkom delovanju sa visokim temperaturama i malom količinom padavina u značajnom delu ispitivane vegetacijske sezone su uticali na funkcionisanje ispitivanih vrsta biljaka na urbanim staništima. Analiza sadržaja hemijskih elemenata u četinama i listovima je pokazala da postoje razlike u sadržaju elemenata između vrsta, ali i da su ispitivane vrste izložene toksičnim koncentracijama B i Sr i istovremenom deficitu esencijalnih elemenata Cu, Mn i Zn. Generalno, kod P. nigra, A. hippocastanum i P. acerifolia postoje razlike u fotosintetičkoj efikasnosti, sadržaju pigmenata i u morfološkim oštećenjima listova. Uprkos navedenom, sve ispitivane vrste, pre svega listopadne A. hippocastanum i P. acerifolia su pokazale visok potencijal za toleranciju stresa zagađivanja toksičnim metalima i metaloidima, što ih čini pogodnim za sadnju u urbanim parkovima i industrijskim zonama, pa kao takve predstavlјaju značajan resurs za dalјa istraživanja.Urban habitats are under strong anthropogenic influence and are often characterized by intense air, water and soil pollution originating from various stationary and mobile sources. The specific type of pollutants in these habitats are metals that due to their toxic, persistent and nonbiodegradable properties accumulate in the soil. Research was conducted in urban parks in Pančevo, Smederevo, Obrenovac and Belgrade that are exposed to pollution by chemical elements, including toxic metals originating from various sources. Research included plant and soil analyses. For ecophysiological research, three woody plant species that have been successfully grown in urban areas were selected: Pinus nigra Arn., Aesculus hippocastanum L. i Platanus acerifolia (Ait.) Willd. The central aim and subject of this doctoral dissertation was to define the factors that limit the functioning of plants, and to determine their ecophysiological characteristics and adaptive responses. Special attention was paid to the problem of pollution by toxic metals and metalloids, their mobility and availability to plants, as well as their impact on physiological processes in plants (photosynthesis efficiency and photosynthetic pigment content). Analyses of the physical and chemical properties of the soils showed that urban soils are characterized by high proportion of a sand fraction, a low content of hygroscopic moisture, organic carbon and nitrogen and an alkaline reaction, but also a favorable C/N ratio for mineralization of organic matter and release of essential minerals for plants. The limiting factors (toxic levels of B, Cr, Ni and Pb measured in soils from urban parks) in synergy with high temperatures and low precipitation during a significant part of the vegetation season, influenced the functioning of the examined plant species in urban habitats. Analysis of the chemical element contents in pine needles and leaves revealed species differences, and also that the examined species were exposed to toxic concentrations of B and Sr and, at the same time, deficits of the essential elements, Cu, Mn and Zn. Generally, in P. nigra, A. hippocastanum and P. acerifolia, differences in photosynthetic efficiency, pigment contents and in morphological symptoms of injury were observed. In spite of the above, all the investigated species, first of all A. hippocastanum and P. acerifolia, exhibited a high potential for tolerance to stress pollution by toxic metals and metalloids, which makes them suitable for planting in urban parks and industrial zones. These species represent a significant resource for further research.

Sensitivity assessment of in vitro and in vivo tests in ecogenotoxicology and guidelines formulation for their application in studies of large lowland rivers

Uvođenje metoda uslovljenim efektima (eng. effect based methods), uključujući i genotoksikološke metode, u programe monitoringa i naučna istraživanja na velikim ravničarskim rekama, omogućilo bi sveobuhvatniju procenu stanja ekosistema i odnosa između samog hemijskog i ekološkog statusa. Osnovni ciljevi disertacije su ispitivanje osetljivosti in vitro i in vivo testova u ekogenotoksikologiji i formulisanje smernica za njihovu primenu u istraživanjima velikih ravničarskih reka. Studija je obuhvatila ukupno 17 lokaliteta u slivovima Velike Morave, Save i Dunava. Za procenu genotoksičnog efekta nativnih i koncentrisanih uzoraka rečne vode, in vitro pristup obuhvatio je SOS/umuC test, alkalni komet test na HepG2 i ZFL ćelijama, kao i analizu ćelijskog ciklusa na ZFL ćelijama. In vivo pristup je za procenu genotoksičnosti obuhvatio alkalni, Fpg modifikovani komet test, kao i mikronukleus test na ćelijama krvi Alburnus alburnus (uklija). Dodatno, ispitivana je osetljivost RAPD analize u detekciji genotoksičnog efekta. U in silico pristupu na osnovu literature analiziran je toksični/genotoksični potencijal hemijskih parametara koji su detektovani u vodi i u uklijama, a integrativnim metodama vršena je kvantifikacija pritiska zagađenja. Biotestovi su pokazali različitu osetljivost u detekciji genotoksičnog efekta. U in vitro istraživanju koncentrisani uzorci rečne vode su se pokazali pogodnim za testiranje toksičnosti/genotoksičnosti, a ZFL ćelije kao dobar model sistem. U in vivo istraživanju A. alburnus se pokazao kao pouzdan bioindikator. Najveća osetljivost u detekciji genotoksičnog efekta u ćelijama krvi dobijena je u alkalnom komet testu. In silico analizom ne može se izvršiti potpuna predikcija genotoksičnog efekta ksenobiotika na organizme.The implementation of effect based methods, including genotoxicological methods, in monitoring and research programs on large lowland rivers, could provide comprehensive assessment of ecosystem quality and relationship between chemical and ecological status itself. The main objectives of the dissertation are examination the sensitivity of in vitro and in vivo tests in ecogenotoxicology and guidelines formulation for their application in studies of large lowland rivers. The study included a total of 17 sites in the Velika Morava, Sava and Danube River basins. To assess the genotoxic effect of native and concentrated river water samples, the in vitro approach included the SOS/umuC test, alkaline comet test on HepG2 and ZFL cells, and cell cycle analysis on ZFL cells. The in vivo approach for genotoxicity assessment included the alkaline, Fpg modified comet and micronucleus test on Alburnus alburnus (bleak) blood cells. Also, the sensitivity of RAPD analysis in the detection of genotoxic effects was examined. Within in silico approach, based on the literature, the toxic/genotoxic potential of chemical parameters detected in water and bleaks was analyzed while the quantification of pollution pressure was performed by integrative methods. Bioassays showed different sensitivity in detecting the genotoxic effect. Within in vitro study, concentrated river water samples proved to be a suitable for toxicity/genotoxicity testing, and ZFL cells a good model system. Within in vivo research, bleak proved to be a reliable bioindicator. The highest sensitivity in the detection of genotoxic effect in blood cells was obtained in the alkaline comet test. A complete prediction of the genotoxic effect of xenobiotics on organisms cannot be performed by in silico analysis.

Allelopathic potential of apple hairy root secondary metabolites - the effect of phloretin and phloridzin in in vitro culture

Ograniĉenja u izuĉavanju potencijala i mehanizama alelopatskog delovanja glavnih sekundarnih metabolita korena jabuke, floretina i florizina, usled kompleksnosti interakcija u rizosferi, mogu biti prevaziĊena upotrebom kulture transformisanih korenova in vitro. Odabranim naĉinom inokulacije regenerisanih izdanaka jabuke sa Agrobacterium rhizogenes 15834, efikasno su indukovani transformisani korenovi kod sorti Melrouz, Zlatni Delińes, Ĉadel i Gloster. Smenom hranljivih podloga sa i bez auksina, po prvi put je uspostavljena permanentna in vitro kultura izolovanih transformisanih korenova jabuke. UHPLC/(+/-)HESI–MS/MS analizom je utvrĊeno da genetiĉka transformacija nije dovela do promena u sadrņaju glavnih sekundarnih metabolita korena jabuke. U eksudatu transformisanih korenova, sadrņanom u teĉnoj podlozi u kojoj su korenovi gajeni, kvantifikovani su iskljuĉivo florizin, floretin, hlorogena i kafeinska kiselina. Tretman ovim eksudatom je prouzrokovao znaĉajnu inhibiciju rastenja i razvića izdanaka i korenova klijanaca Arabidopsis thaliana. Sliĉan inhibitorni efekat ispoljili su ĉist florizin u koncentraciji od 500 μM, kao i floretin u svim testiranim koncentracijama (125-500 μM), izazivajući krupne promene u morfologiji tretiranih klijanaca, posebno korena. Velike sliĉnosti su zapaņene u obrascu promena ekspresije gena ukljuĉenih u polarni transport auksina, kao i biosintezu auksina i giberelina u korenovima klijanaca arabidopsisa tretiranih eksudatom transformisanih korenova jabuke, sa onim detektovanim kod klijanaca tretiranih 500 μM floretinom. To ukazuje na mogućnost da je floretin u eksudatu korena jabuke metabolit koji je odgovoran za alelopatske efekte, a koji bi se mogli zasnivati na promenama nivoa auksina i giberelina, kao i na interakciji ove dve grupe hormona.The investigation of allelopathic potential and mode of action of the main apple root secondary metabolites, phloridzin and phloretin is difficult to perform due to high complexity of rhizosphere interactions. Therefore, the generation of in vitro hairy root cultures could be an alternative system for allelopathic studies of apple. The efficient induction of hairy roots in apple cvs. Melrose, Golden Delicious, Ĉadel and Gloster was obtained using the appropriate mode of inoculation of regenerated shoots by Agrobacterium rhizogenes 15834. Permanent in vitro culture of apple autonomous hairy roots was established for the first time by cultivation on alternating growth media with and without auxins. UHPLC(+/-)HESI-MS/MS analysis confirmed that genetic transformation did not lead to changes in the content of the main secondary metabolites of apple roots. Dihydrochalcones phloridzin and phloretin, chlorogenic and caffeic acids were detected as only putative allelochemicals exuded into the growth medium in which hairy roots were grown. Apple hairy root exudates significantly affected development and growth of Arabidopsis thaliana seedlings. Pure phloridzin at 500 μM and phloretin in all tested concentrations (125-500 μM) had similar effects as apple hairy root exudates inducing significant alterations in morphology of seedlings, expecially roots. High similarities in the expression patterns of genes involved in polar auxin transport and auxin and gibberellin biosynthetic pathways in roots of arabidopsis seedlings treated with either apple hairy root exudates or 500 μM phloretin were observed. This indicates that phloretin in hairy root exudates could be at least partly responsible for the apple root allelopathic properties, that were most likely based on auxin and gibberellin cross-talk and their altered homeostasis in arabidopsis roots.

Molekularna struktura, poreklo i funkcija B hromozoma kod žutogrlog šumskog miša Apodemus flavicollis (Mammalia, Rodentia)

B hromozomi predstavljaju prekobrojne hromozome koji nisu neophodni za normalan rast i razviće. Ovi prekobrojni hromozomi se javljaju kod svih višećelijskih eukariotskih grupa, osim kod ptica, i različiti su po poreklu, morfologiji, veličini, broju, genetičkoj strukturi i efektima na fenotip. Njihova prisutnost u živom svetu procenjuje se na 3% kariotipski analiziranih vrsta. Karakteriše ih nasleđivanje koje ne prati Mendelova pravila, kao i izostanak rekombinacije sa hromozomima osnovnog A seta. Glavna pitanja koja se postavljaju u savremenim istraživanjima B hromozoma se odnose na njihovo poreklo, mehanizme nastanka i održavanja. Poznavanje genetičkog sadržaja, transkripcionog statusa i efekata prekobrojnih hromozoma, omogućuje rasvetljavanje njihovog biološkog značaja. Glodari su najbrojniji nosioci B hromozoma među sisarima. Rod Apodemus broji ukupno 20 vrsta, od kojih je kod šest zabeleženo prisustvo B hromozoma. U ovoj studiji izučavani su B hromozomi na jedinkama vrste žutogrli miš, Apodemus flavicollis. Nosioci imaju najčešće jedan ili dva B, dok je maksimalan zabeležen broj osam B hromozoma. Prekobrojni hromozomi se kod ove vrste javljaju u svim do sada proučavanim populacijama na teritoriji Republike Srbije, sa različitom učestalošću. Brojne populacione studije na ovoj vrsti pokazuju da jedinke sa B hromozomima imaju višu adaptivnu vrednost u poređenju sa jedinkama bez prekobrojnih hromozoma. Cilj ove studije bio je utvrđivanje sekvence, proučavanje porekla, kao i analiza transkripcione aktivnosti pojedinih sekvenci B hromozoma vrste A. flavicollis. U istraživanje su uključene životinje sa lokaliteta u Republici Srbiji, Ruskoj federaciji i Republici Belorusiji. Metodom mikrodisekcije iz kariotipa tri jedinke su izolovani B hromozomi koji su potom umnoženi u lančanoj reakciji polimeraze (PCR). Proizvedene su fluorescentne B-specifične probe. Procena homologije između B-specifičnih proba i hromozoma osnovnog kariotipa iste vrste izvršena je putem fluorescentne in situ hibridizacije (FISH). Utvrđena je homologija između sekvence B hromozoma i: a) pericentromernog regiona (PR) polnih hromozoma, b) subtelomernih regiona dva para malih autozoma, kao i c) slabija homologija sa distalnim regionom Y hromozoma. Homologija između sekvenci različitih B hromozoma je potvrđena bez obzira na geografsku udaljenost populacija iz kojih uzorci vode poreklo i nezavisno od broja B hromozoma u kariotipu. Ovo ukazuje na isto poreklo i/ili strukturu svih B hromozoma kod vrste A. flavicollis. U ovoj studiji je takođe pokazano da i pored česte zastupljenosti C-kit gena na B hromozomima različitih vrsta sisara, ovaj gen nije pronađen na B hromozomima vrste A. flavicollis. Poređenjem sekvenci B hromozoma sa referentnim genomom vrste Mus musculus, identifikovano je 39 gena sa 22 hromozomska regiona koja vode poreklo sa 16 hromozoma referentnog genoma. Sa izuzetkom B hromozoma poreklom iz Ruske federacije, koji u sekvenci sadrži jedan dodatni hromozomski region koji nije nađen kod populacija iz Republike Srbije, rezultati pokazuju da su svi analizirani B hromozomi izgrađeni od istih regiona hromozoma A seta. Sekvenca B hromozoma je u najvećoj meri sadrži gene koji kodiraju proteine mikrotubula i ćelijskog ciklusa. Takođe su prisutni i geni koji kodiraju nukleotid-vezujuće, metal-vezujuće proteine kao i proteine membrane, identifikovan je i veliki broj repetitivnih sekvenci, među kojima su transpozoni u nekim slučajevima bili tandemski organizovani. Utvrđeno je i da se geni smešteni na B hromozomima nalaze u različitim stadijumima degradacije. Nasuprot višedecenijskom uverenju da su B hromozomi genetički inertni, u ovom istraživanju je pokazano da se pseudogeni poreklom sa B hromozoma transkribuju. Pored toga, neizmenjeni geni sa B hromozoma, ili sa osnovnog genoma, u prisustvu B hromozoma, pokazuju viši nivo transkripcije kod ženki u mlađem životnom dobu. Transkripcija gena Cenpe je povezana i sa brojem B hromozoma, tako da je nivo transkripcije najviši kod jedinki koje imaju jedan B hromozom. B hromozomi vrste A. flavicollis, najverovatnije vode poreklo od pericentromernog regiona X hromozoma koji se nalazi neposredno uz pseudoautozomalni region, odlikuju se stabilnom genetičkom strukturom na širokom arealu vrste, transkripciono su aktivni na način koji je povezan sa polom, starošću i brojem B hromozoma u kariotipu.B chromosomes (Bs) are supernumerary chromosomes in a standard karyotype, unnecessary for normal growth and development. Bs are present in all main groups of multicellular eukaryotes, except birds, and extremely variable considering origin, morphology, size, number, molecular structure, and phenotypic effects. 3% of all analyzed species have B chromosomes. The Bs do not follow rules of Mendelian segregation law and do not recombine with members of the basic A chromosome complement. The main questions considered in contemporary studies of Bs are surrounding their origin, mechanisms of formation and maintenance. Knowledge of their molecular structure, transcriptional activity, and phenotypic effects, will allow a deeper understanding of their biological importance. Within mammals, the most frequent carriers of Bs are rodents. The genus Apodemus includes 20 species, six of which carry Bs, in addition to A chromosome set. In this study, B chromosomes were analyzed in specimens of the yellow-necked mouse, Apodemus flavicollis. Carriers usually have one or two Bs, and the highest recorded number in this species is eight B chromosomes. In different frequencies, supernumeraries are present in all studied populations of A. flavicollis in the territory of the Republic of Serbia. Numerous population studies of this species are indicating that B carriers have a higher fitness compared to individuals without B chromosomes. The aim of this study was to define sequence, origin and transcriptional activity of selected B regions in species A. flavicollis. Specimens were collected from different localities in the Republic of Serbia, Russian Federation and the Republic of Belarus. B chromosomes have been isolated by microdissection from the karyotype of three individuals and amplified in the polymerase chain reaction (PCR). B-specific fluorescent probes were synthesized. Estimation of homology between B-specific probes and chromosomes of A set had been obtained by fluorescent in situ hybridization (FISH). Homology was shown between the B chromosome and: a) pericentromeric region (PR) of sex chromosomes b) subtelomeric region of the two pairs of small chromosomes and c) lower homology with the distal region of the Y chromosome. Despite the distinct geographical origin of samples and different number of Bs in karyotypes, hybridized probes showed the same affinity to Bs. This indicates the same origin and/or structure of all Bs in A. flavicollis. This study has demonstrated that C-kit gene does not contribute to the composition of supernumeraries in A. flavicollis, contrary to the studies of Bs in numerous mammalian species. Comparing sequences of Bs and referent genome of the species Mus musculus, 39 genes in 22 chromosomal regions that originate from 16 chromosomes of the referent genome have been identified. With the exception of one region present on B chromosome from Russian Federation, that contained one additional chromosomal region not present on the samples from Serbian populations, results demonstrate that all analyzed Bs are composed of the same A-chromosomes’ regions. B chromosomes are rich in microtubule and cell cycle protein-coding genes. Other present categories include genes that are coding nucleotide-binding, membrane, and metal-binding proteins, as well as a high amount of repetitive elements. Transposable elements were tandemly arranged in some cases. Genes present on B chromosomes are at different stages of degradation. In contrast to the view accepted for many decades that Bs are genetically inert elements, this study demonstrates that pseudogenes on Bs are transcribed. The study also demonstrates that Bs-genes, or standard-genome genes in the presence of Bs, show higher transcriptional levels in young females. Furthermore, transcription of Cenpe gene is linked with the number of Bs, in the manner that samples with one B show the highest transcription level. B chromosomes in A. flavicollis probably originate from the pericentromeric region of the X chromosome which is positioned near pseudoautosomal region, they are featured with stable genetic structure throughout the whole areal of the species and are transcriptionally active depending on the sex, age and number of Bs.

The effects of propofol induced anesthesia on synaptic plasticity, dopaminergic system activity and behaviour of juvenile rats

Propofol je često korišćeni anestetik u modernoj medicini. Adiktivni potencijal propofola je uočen, kao i uticaj na memorijski proces. Međutim, oba fenomena su još uvek nedovoljno istražena. Imajući u vidu da je adolescencija period izuzetne osetljivosti na dejstvo adiktivnih supstanci i intenzivne maturacije mnemoničkog potencijala, cilj ovog rada je bio da se ispita uticaj jednokratnog izlaganja propofolskoj anesteziji, što je tipičan način kliničke primene anestetika, na dopaminsku signalizaciju, sinaptičku i neuronsku aktivnost u različitim regionima mozga i ponašanje juvenilnih/peripubertetskih pacova, kao model sistema humanog periadolescentnog razvoja. Efekti su analizirani 4, 24 i 48 sati nakon tretamana, kod mužjaka Wistar pacova starih 35 dana. Dobijeni rezultati su po prvi put ukazali da izlaganje propofolskoj anesteziji izaziva promene u ekspresiji/fosforilaciji signalnih molekula koji su već prepoznati kao značajni za dejstvo adiktivnih supstanci. Od svih analiziranih dopaminoceptivnih moždanih regiona (medijalni prefrontalni korteks, strijatum i talamus) jedino su u talamusu uočene značajne promene u ekspresiji fosforilisane/aktivirane forme DARPP-32 proteina, pokazatelja postsinaptičke dopaminske signalizacije, 4 i 24 sata nakon tretmana, i bile su praćene povećanom ekspresijom FosB/ΔFosB proteina, biohemijskog pokazatelja neuronske aktivnosti. Promene su lokalizovane u paraventrikularnom talamičkom jedru i mediodorzalnom talamusu. U strijatumu i medijalnom prefrontalnom korteksu je uočen porast u ekspresiji fosforilisane forme CaMKIIα, biohemijskog senzora sinaptičke aktivnosti koji ima važnu ulogu u pamćenju prethodnog izlaganja adiktivnim supstancama. Smanjenje u intenzitetu anksioznosti (procenjeno na osnovu rezultata dobijenih u testu svetlo/tamne kutije i uzdignutog krstastog lavirinta) je zabeleženo 24 sata nakon tretmana, kada je uočen i pad u ekspresiji FosB proteina u strijatumu, što se može tumačiti kao traženje senzacija usled smanjene aktivnosti moždanog regiona značajnog za osećaj zadovoljstva i motivisanost. Pojačan motorički odgovor na d-amfetamin i fenciklidin je uočen 24 sata nakon tretmana (ukrštena senzitizacija), kao potvrda da bez obzira na različite primarne mehanizme dejstva propofol i dve korišćene droge koriste iste neuronske puteve za ostvarivanje psihomotoričkih efekata. Rezultati studije su takođe ukazali da kod peripubertetskih pacova nakon izlaganja propofolskoj anesteziji postoje izvesne poteškoće u pozivanju memorije i memorisanju novih informacija u neaverzivnim memorijskim testovima (prostorna habituacija, prepoznavanje novog objekta), 24 sata nakon tretmana. Ove posledice su bile praćene promenama u ekspresiji molekula koji učestvuju u pozivanju i rekonsolidaciji epizodične memorije (BDNF/TrkB, Egr-1, ERK1/2, CaMKIIα, FosB/ΔFosB), u dorzalnom hipokampusu. Uočene poteškoće u prepoznavanju novine su u skladu sa malobrojnim kliničkim studijama, koje naglašavaju značaj kontrole osnovnih kognitivnih funkcija kod starije dece nakon izlaganja propofolskoj anesteziji. Takođe, u svim ispitivanim moždanim regionima osim u talamusu je zabeležen porast u ekspresiji fosforilisane forme Aducinβ proteina, što je indikacija za destabilizaciju citoskeleta i sinaptičku reorganizaciju. Gledano u celini, rezultati ove studije su ukazali da bolje razumevanje bioloških posledica primene propofola u juvenilnom/pubertetskom uzrastu može doprineti sagledavanju starosno zavisnih zdravstvenih rizika tretmana, koji su do sada intenzivno istraživani samo u ekstremima životne dobi, tj. u ranoj postnatalnoj i kasnoj životnoj fazi.Propofol is a commonly used anesthetic in modern medicine. Addictive potential of propofol is observed, as well as the impact on the memory process. However, both phenomena are still insufficiently explored. Bearing in mind that adolescence is a period of extreme sensitivity to addictive substances and intense maturation of the mnemonic potential, the aim of this study was to examine the effect of a single exposure to propofol anesthesia, which is a typical method of its clinical application, on dopaminergic signaling, synaptic and neuronal activity in different brain regions and behavior of juvenile/peripubertal rats, as a model system of human periadolescencent development. The effects were analyzed 4, 24 and 48 hours after the treatment, in male Wistar rats aged 35 days. The obtained findings for the first time showed that exposure to propofol anesthesia caused changes in the expression/phosphorylation of signal molecules that are already recognized as significant for the action of the addictive substances. Of all the analyzed dopaminoceptive brain regions (medial prefrontal cortex, striatum and thalamus), significant changes in the expression of the phosphorylated/activated form of DARPP-32 protein, indicator of postsynaptic dopaminergic signalling, were observed only in the thalamus, 4 and 24 hours after the treatment, and were accompanied by increased expression of FosB/ΔFosB protein, a biochemical indicator of neuronal activity. The alterations were localized in the paraventricular thalamic nucleus and the mid-dorsal thalamus. An increase in the expression of the phosphorylated form of CaMKIIα, a biochemical sensor of synaptic activity that has an important role in memory on addictive substances exposure, was detected in striatum and medial prefrontal cortex. Reduction in the intensity of anxiety (estimated in accordence to the data obtained in the light/dark box and elevated plus maze tests) was observed 24 hours after the treatment, along with the decrease in the expression of FosB protein in striatum, which can be interpreted as a sensation seeking due to decreased activity of brain region important for a pleasure/motivation. An enhanced motor response to d-amphetamine and phencyclidine was observed 24 hours after the treatment (cross sensitization), as confirmation that despite the different primary mechanisms of action, propofol and these two drugs use the same neuronal pathways to achieve psychomotor effects. The results of the study also indicated that in peripubertal rats exposed to propofol anesthesia, there were some difficulties in memory retrieval and acquisition of new learning in non-aversive memory tests (spatial habituation, novel object recognition), 24 hours after the treatment. These effects were accompanied by changes in the expression of molecules involved in retrieval and reconsolidation of episodic memory (BDNF/TrkB, Egr-1, ERK1/2, CaMKIIα, FosB/ΔFosB) in the dorsal hippocampus. Detected difficulties in recognizing the novelty are consistent with a few clinical studies that emphasize the importance of controlling basic cognitive functions in older children after exposure to propofol anesthesia. Also, in all investigated brain regions, except the thalamus, an increase in the expression of the phosphorylated form of Adducinβ protein was observed, which is an indication of the cytoskelet destabilization and synaptic reorganization. Overall, the results of this study accentuated that a better understanding of the biological consequences of the use of propofol in juvenile/puberty period could contribute to the undestanding of age-dependent health risks of the treatment, which have been extensively investigated so far only at the extremes of age, i.e. during early postnatal period and in aging.

The chemical properties of potentially toxic elements in soil and their impact on the functioning of the species Betula pendula Roth., Acer pseudoplatanus L. and Acer platanoides L. in urban areas of several industrial centres in Serbia

Staništa u urbanoj sredini i u industrijskim zonama izločena su hroničnom zagađivanju poreklom iz različitih mobilnih i stacionarnih izvora što kao rezultat moče imati kontaminaciju vazduha, zemljišta i voda, a u kombinaciji sa specifičnom urbanom klimom uključujuši i „gradska ostrva toplote“ predstavlja izvor stresa za biljke. Poseban vid zagađenja u urbanim sredinama predstavlja zagađenje toksičnim elementima čiji je sadrčaj u urbanim zemljištima promenjiv, a njihova mobilnost i biodostupnost zavisi od oblika i asocijacije sa različitim fazama zemljišta. Biljke koje opstaju na urbanim staništima pokazuju različite strukturne i funkcionalne simptome oštešenja u zavisnosti od tipa i intenziteta zagađenja i mogu se koristiti kao biomonitori i/ili bioindikatori zagađenja. U skladu sa tim, osnovni cilj i predmet ove doktorske disertacije je bio da se utvrdi sadrčaj, biodostupnost i mobilnosti hemijskih elemenata u urbanim zemljištima, njihova akumulacija u različitim delovima biljaka, kao i njihov uticaj na funkcionisanje biljaka na urbanim staništima. Poligoni za istračivanja su bili urbani parkovi, izločeni različitim izvorima zagađenja u: Pančevu, Smederevu, Obrenovcu i Beogradu, a kao model vrste koriššene su tri drvenaste vrste: Acer platanoides L., Acer pseudoplatanus L. i Betula pendula Roth. Analizirane su osnovne fizičko - hemijske karakteristike zemljišta, i sadrčaj hemijskih elemenata u zemljištu i biljnom materijalu. Uzorci zemljišta i biljnog materijala pripremeljeni su vlačnom digestijom u mikrotalasnom digestoru pomošu azotne kiseline i vodonik peroksida, a uzorci zemljišta su podvrgnuti i modifikovanoj BCR sekvencijalnoj ekstrakciji. Nakon ekstrakcije, u rastvorima je određivana koncentracija Al, As, B, Cd, Cr, Cu, Fe, Li, Mn, Ni, Pb, Se, Sr i Zn metodom induktivno spregnute plazme sa optičko emisionom spektrometrijom (ICP-OES). Analiziran je fotosintetički odgovor biljaka na efekte potencijalno toksičnih elemenata merenjem efikasnosti fotosinteze i koncentracije fotosintetskih pigmenata ispitivanih vrsta i detektovani su i opisani morfološki simptomi oštešenja listova i stanja struktura periferijskih zaštita listova uključujuši i hemijsku analizu atmosferskih čestica deponovanih na spoljašne površine listova. Rezultati su statistički obrađeni koriššenjem korelacione analize, analize glavnih komponenti, diskriminantne analize i faktorijalne analize varijansi. Utvrđeno je da analizirana zemljišta pripadaju klasi peskovito-glinovite ilovače i glinovite ilovače, da ih odlikuje mali procenat vlage, organske materije i alkalna reakcija koja predstavlja glavni limitirajuši faktor za usvajanje elemenata od strane biljaka, pri čemu je sadrčaj B, Cr, Ni i Pb u pojedinim parkovima viši od maksimalno dozvoljenih koncentracija saglasno pravilniku Republike Srbije (SG RS 23/94). Utvrđeno je takođe da ispitivani elementi uglavnom vode poreklo iz matičnog supstrata, ali i da na njihov sadrčaj utiču antropogeni izvori zagađenja i specifičan način formiranja urbanih zemljišta. Antropogeni uticaj je najizračeniji u parkovima u Smederevu, Obrenovcu i Beogradu. Sekvencijalnom analizom uzoraka zemljišta utvrđeno je da najmanju mobilnost u ispitivanim zemljištima imaju Al, Cr i Fe, ukazujuši na njihovu snačnu strukturnu povezanost sa primarnim i sekundarnim silikatnim mineralima zemljišta i njihovu slabu dostupnost biljkama. Cu, Li, Ni i Pb su slabo mobilni i veoma stabilni elementi, dok se Zn i B svrstavaju u grupu srednje mobilnih elemenata. Mangan i Sr odlikuje najveša mobilnost i potencijalna dostupnost biljkama. Merenje i analiza akumulacije potencijalno toksičnih elemenata u listovima i kori, efikasnosti fotosinteze i sadrčaja fotosintetičkih pigmenata su pokazale da ispitivane vrste generalno pokazuju malu osetljivost na stresne efekte akumuliranih elemenataali i da postoje razlike specifične za vrstu. Na osnovu analiziranih morfo - fizioloških parametara ispitivanih biljaka, utvrđena je određena gradacija u smislu tolerantnosti na efekte potencijalno toksičnih elemenata: breza > mleč > javor. Imajuši u vidu sve navedeno, jasno je da ispitivane vrste ne mogu biti dobar izbor za bioindikaciju i/ili biomonitoring kvaliteta urbane sredine, izuzev u slučaju Sr gde bi eventualno mogle da se primene kao bioindikatori.Habitats in the urban environment and industrial zones are exposed to chronic pollution originating from various mobile and stationary sources, which can result in air, soil and water contamination. All of these, combined with the specific urban climate, including "urban heat islands", are a source of stress for plants. A special type of pollution in urban areas is caused by toxic elements; their content in urban soils varies and their mobility and bioavailability depend on forms and associations with different soil phases. Plants that survive in the urban environment exhibit different structural and functional damage symptoms depending on pollution type and intensity and can be used as biomonitors and/or bioindicators of pollution. Accordingly, the main aim and subject of this doctoral dissertation was to determine the content, bioavailability and mobility of chemical elements in urban soils, their accumulation in different plant parts, and their impact on the functioning of plants in urban habitats. The study area comprised urban parks exposed to various pollution sources in Panţevo, Smederevo, Obrenovac and Belgrade, while three tree species were used: Acer platanoides L., Acer pseudoplatanus L. and Betula pendula Roth. Selected physical and chemical soil parameters and chemical element content in soil and plant material were analysed. Soil samples and plant material were digested in a microwave, using nitric acid and hydrogen peroxide, and the soil samples were extracted according to a modified BCR sequential method. After extraction, Al, As, B, Cd, Cr, Cu, Fe, Li, Mn, Ni, Pb, Se, Sr and Zn concentrations were measured by inductively coupled plasma optical emission spectrometry (ICP-OES). The photosynthetic response of the plants to the effects of potentially toxic elements was analysed by measuring photosynthetic efficiency and photosynthetic pigment content in the investigated species. In addition, morphological symptoms of leaf damage and the condition of the peripheral leaf protection structures were also described, including a chemical analysis of atmospheric particles deposited on the outer surface of the leaves. The results were statistically analysed using correlation analysis, principal component analysis, discriminant analysis, and factorial analyses of variance. It was found that the analysed soils belonged to the class of sandy clay loam and clay loam, characterised a low percentage of moisture and organic matter, and an alkaline reaction, which was the main limiting factor for the absorption of elements by plants, whereby B, Cr, Ni and Pb content in some parks was higher than the maximum allowed concentration in accordance with the regulations of the Republic of Serbia (SG RS 23/94). Moreover, it was found that the investigated elements originated mainly from the parent rock, but that their content was also influenced by anthropogenic sources of pollution and the specific way urban soils are formed. The anthropogenic impact was most pronounced in the parks in Smederevo, Obrenovac and Belgrade. Sequential analysis of soil samples found that Al, Cr and Fe exhibited the lowest mobility at the investigated sites, indicating their strong structural connection with primary and secondary silicate soil minerals and their low availability to plants. Cu, Li, Ni and Pb exhibited poor mobility and were very stable elements, while Zn and B were classified as elements with medium mobility. Manganese and Sr were characterised by the highest level of mobility and potential availability in plants. The measurement and analysis of the accumulation of potentially toxic elements in leaves and bark, photosynthetic efficiency and photosynthetic pigment content revealed that the tested species generally displayed low sensitivity to the stress effects of the accumulated elements and that there were species-specific differences. Based on the analysed morpho - physiological parameters of the investigated plants, a certain gradation was determined in terms of tolerance to the effects of potentially toxic elements: birch > Norway maple > sycamore maple. Bearing in mind all the above, it is clear that the examined species are not good choices for bioindication and/or the biomonitoring of pollution in urban environments, except in the case of Sr, when they could possibly be used as bioindicators.