IMMUNOGLOBULIN G GLYCOSYLATION IN DOWN SYNDROME

Glikozilacija je jedna od najčešćih ko- i posttranslacijskih modifikacija proteina. Glikani vezani na imunoglobulin G (IgG) utječu na njegovu strukturu i funkciju i mijenjaju se s dobi te u brojnim fiziološkim i patološkim stanjima organizma. Znakovi preuranjenog starenja, kao i brojne druge bolesti, česti su kod Downova sindroma (DS). Tekućinskom kromatografijom ultravisoke učinkovitosti analizirana je glikozilacija IgG-a u tri populacije osoba s DS-om za koje su postojali klinički podaci o najčešćim komorbiditetima i uspoređena s onom njihovih zdravih vršnjaka. Proučen je utjecaj produkata gena kromosoma 21 B3GALT5, RUNX1 i DYRK1A na glikozilaciju IgG-a u DS-u digestijom glikana IgG-a egzoglikozidazama te kemijskom inhibicijom produkata gena u limfoblastoidnim staničnim linijama. Pokazano je kako glikozilacija IgG-a osoba s DS-om neovisno o učestalim komorbiditetima odgovara kronološki starijim zdravim osobama. U korištenim eksperimentalnim sustavima RUNX1 ima izravan utjecaj na glikozilaciju IgG-a u DS-u, dok B3GALT5 i DYRK1A nemaju.Glycosylation is one of the most common co- and posttranslational modifications. Glycans attached to immunoglobulin G (IgG) affect its function and change with age and in numerous physiological and pathological conditions. Signs of premature aging, along with many other diseases, are common in Down syndrome (DS). IgG glycosylation of three DS populations that had clinical data on the most common comorbidities was analyzed by ultra-high performance liquid chromatography and compared to their healthy peers. The impact of chromosome 21 gene products B3GALT5, DYRK1A and RUNX1 on IgG glycosylation was studied using enzymatic digestions of IgG glycans and IgG-producing lymphoblastoid cell lines obtained from individuals with and without DS. IgG glycosylation was revealed to be significantly shifted in the direction of aging in DS, showing a ubiquitous trend independent of comorbidities, and an insight into the effect of gene products B3GALT5, DYRK1A and RUNX1 on IgG glycosylation was provided

Položaj kromosoma 1 i 9, obilježenih pomoću tehnologije CRISPR/Cas9, u odnosu na jezgrinu ovojnicu i centrosome

Human chromosomes occupy specific territories in the cell nucleus. Chromosomes that are localized at the periphery of the cell nucleus at the onset of mitosis show increased missegregation rates, in compromised mitosis conditions, than the chromosomes that are localized centrally. I specifically labeled subtelomeric region of chromosome 1 and pericentromeric region of chromosome 9 using technology CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9). I determined their position in the cell nucleus, during early prometaphase, with respect to the nuclear envelope and centrosomes. Chromosome 1 is localized at the periphery, whereas chromosome 9 is equally distributed in the cell nucleus during early prometaphase. Furthermore, chromosome 1 and chromosome 9 are both equally polar (closing the angle of >90º with the spindle pole) during early prometaphase. Also, there is no difference in the distance between chromosome 1 and chromosome 9 from the centrosome in early prometaphase and the average distance of the centrosome to the former nuclear envelope in early prometaphase is 2.79 μm. The position of centrosomes during early prometaphase is important from the point of mitotic fidelity. The results of this thesis further support future research of specific chromosome position during early prometaphase.Kromosomi ljudskih stanica zauzimaju specifične teritorije u jezgri. Kromosomi koji se nalaze na periferiji jezgre prije početka mitoze skloniji su pogrešnoj podjeli, u uvjetima narušene mitoze, u odnosu na kromosome koji su pozicionirani centralno. Specifično sam obilježila subtelomernu regiju kromosoma 1 i pericentromernu regiju kromosoma 9 upotrebom tehnologije CRISPR/Cas9 (engl. clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9). Odredila sam položaj navedenih kromosoma u jezgri, tijekom rane prometafaze, u odnosu na jezgrinu ovojnicu i centrosome. Kromosom 1 zauzima periferni položaj, dok je kromosom 9 podjednako distribuiran u svim dijelovima jezgre tijekom rane prometafaze. Nadalje, kromosom 1 i kromosom 9 su podjednako polarni (zatvaraju kut >90º s polom diobenog vretena) tijekom rane prometafaze. Također, ne postoji razlika u udaljenosti kromosoma 1 i kromosoma 9 od centrosoma u ranoj prometafazi te prosječna udaljenost centrosoma od nekadašnje jezgrine ovojnice tijekom rane prometafaze iznosi 2,79 μm. Pozicija centrosoma tijekom rane prometafaze važna je za pravilnu diobu. Rezultati proizašli iz ovoga rada podupiru buduća istraživanja položaja specifičnih kromosoma tijekom rane prometafaze

Inhibition of human cholinesterases by O-alkyloximes

Acetilkolinesteraza (AChE) i butirilkolinesteraza (BChE) su enzimi koji kataliziraju hidrolizu acetilkolina i njihova inhibicija jedna je od glavnih strategija u liječenju Alzheimerove bolesti (AB). No, budući da je AB multifaktorijalna bolest, noviji pristup liječenju je razvoj lijekova s višestrukim učinkom koji bi istovremeno djelovali na više mogućih uzročnika bolesti. U sklopu ovog diplomskog rada testirano je 20 O-alkiloksima kao inhibitora ljudskih AChE i BChE, a za nekolicinu spojeva procijenjena je sposobnost vezanja u periferno mjesto AChE i sposobnost inhibicije β-amiloidne samoagregacije. Svi ispitani spojevi reverzibilno su inhibirali obje kolinesteraze u mikromolarnom području, s konstantama inhibicije Ki u rasponu 0,30 – 360 μmol dm–3. Najjačim inhibitorom AChE pokazao se spoj 20 (Ki = 6 μmol dm–3), dok je BChE najjače inhibirao spoj 15 (Ki = 0,30 μmol dm–3). Gotovo svi ispitivani O-alkiloksimi pokazali su se selektivnim inhibitorima BChE u odnosu na AChE. Sposobnost vezanja u periferno mjesto AChE i moguće sprječavanje nastajanja neurotoksičnog kompleksa AChE-β-amiloid pokazali su spojevi 2, 18, 19 i 20. Kao inhibitori β-amiloidne samoagregacije istaknuli su se spojevi 18 i 20. Za sve spojeve je in silico procijenjena sposobnost prolaska kroz krvno-moždanu barijeru te je pokazano da bi mogli prijeći krvno-moždanu barijeru pasivnim transportom.Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are enzymes that catalyze the hydrolysis of acetylcholine, and their inhibition is one of the main strategies in the treatment of Alzheimer's disease (AD). But, AD is a multifactorial disease so a new approach to treatment is development of multi-target directed ligands that would simultaneously act on several possible causes of the disease. As part of this thesis, 20 O-alkyloximes were tested as inhibitors of human AChE and BChE, and some of them were also tested for ability to bind to the peripheral site of AChE and to inhibit β-amyloid self-aggregation. All tested compounds reversibly inhibited both cholinesterases with inhibition constants Ki in the range of 0,30 – 360 μmol dm–3. The most potent AChE inhibitor was compound 20 (Ki = 6 μmol dm–3), while that of BChE was compound 15 (Ki = 0,30 μmol dm–3). Tested O-alkyloximes were generally more selective inhibitors of BChE than of AChE. Compounds 2, 18, 19 and 20 showed the ability to bind to the peripheral site of AChE and possible attenaution of formation of the neurotoxic AChE-β-amyloid complex. Compounds 18 and 20 proved to inhibit β-amyloid self-aggregation. All compounds were evaluated in silico for ability to pass the blood-brain barrier and all of them should be able to pass the blood-brain barrier by passive transport

Gamma-radiolytic degradation of nabumetone in water

Nabumeton je jedan od nesteroidnih lipofilnih protuupalnih lijekova. Farmakološko djelovanje ostvaruje putem metabolita 6-metoksi-2-naftiloctene kiseline, koji je inhibitor enzima ciklooksigenaze. Međudjelovanjem ionizirajućeg zračenja i tvari nastaju reaktivne kratkoživuće čestice, oksidirajuće i reducirajuće vrste, koje neselektivnim reakcijama reagiraju s farmaceuticima te ih mogu prevesti u za okoliš neškodljive produkte. U sklopu ovog diplomskog rada, za kvantitativno određivanje nabumetona korištena je tekućinska kromatografija visoke djelotvornosti i UV/Vis spektrofotometrija. Djelotvornost razgradnje praćena je u vodenim otopinama pri različitim koncentracijama nabumetona, različitim dozama zračenja te različitim vrijednostima pH. Također, praćen je i utjecaj različitih "hvatača" radikala, anorganskih iona te otapala. Spregnuti sustav tekućinske kromatografije ultravisoke djelotvornosti i spektrometrije masa visokog razlučivanja korišten je za analizu razgradnih produkata nabumetona. Na temelju rezultata analize tandemnom spektrometrijom masa, pretpostavljene su strukture i sheme fragmentacije razgradnih produkata nabumetona.Nabumetone is one of the non-steroidal anti-inflammatory drugs, characterized by its non-acidic and lipophilic properties. Its pharmacological effects are achieved through the metabolite 6-methoxy-2-naphthylacetic acid, which is an inhibitor of the cyclooxygenase enzyme. Interaction of ionizing radiation and the substance results in the formation of reactive short- lived particles, oxidizing and reducing species, which, through non-selective reactions, react with pharmaceuticals and can transform them into environmentally benign products. In this work, high performance liquid chromatography and UV/Vis spectrophotometry were used for the quantification of nabumetone. The degradation efficiency was monitored in aqueous solutions at different initial concentrations of nabumetone, different radiation doses, and different pH values. Additionally, the impact of various radical scavengers, inorganic ions, and solvents was observed. A coupled system of ultra-high-performance liquid chromatography and high-resolution mass spectrometry was used for the analysis of nabumetone degradation products. Based on the results of tandem mass spectrometry analysis, structures and fragmentation schemes of nabumetone degradants were proposed

Targeting the fusion protein RIOX1-dSpCas9 to the promoter region of B4GALT1 for removal of the histone mark H3K4me3

Šest epigenetičkih mehanizama simultanim djelovanjem regulira gensku ekspresiju, a njihove su interakcije, zastupljenosti i efekti na transkripcijski status određenog gena specifični u odnosu na kromatinski „okoliš“. Cilj istraživanja bio je ukloniti histonsku oznaku H3K4me3 u promotorskoj regiji gena B4GALT1 pomoću fuzijskog proteina RIOX1-dSpCas9 navođenog na specifičnu regiju sa šest različitih molekula sgRNA (od engl. single guide RNA). Također, cilj je bio dizajnirati testove za analizu stupnja metilacije pirosekvenciranjem ove regije. Metodom kromatinske precipitacije CUT&RUN, nakon koje slijedi qPCR, utvrđeno je da nije došlo do statistički značajnog smanjenja u količini histonske oznake H3K4me3 kao ni do povećanja stupnja metilacije DNA u analiziranoj regiji. Metodom kvantitativnog PCR je utvrđeno da nije došlo do promjene u genskoj ekspresiji. Gen B4GALT1 ima složenu genetičku i epigenetičku regulaciju, a efekt fuzijskog proteina RIOX1-dSpCas9 mogao je ostati nezapažen zbog uskog područja koje je analizirano, ali i zbog već prisutnih epigenetičkih modifikacija i strukture kromatina koji su mogli onemogućiti njegovo vezanje ili katalitičku aktivnost.Gene expression is regulated by six epigenetic mechanisms that work simultaneously. Their interactions as well as their effects on the transcriptional status of a certain gene are specific and depend on chromatin context. The goal of this research was to remove the histone mark H3K4me3 from the promoter region of the B4GALT1 gene using the fusion protein RIOX1-dSpCas9, which was guided to a specific region by six different sgRNA molecules. Also, the goal was to design assays for pyrosequencing in order to analyze DNA methylation level in this region. The CUT&RUN chromatin precipitation method followed with qPCR revealed no statistically significant change in the amount of histone mark H3K4me3 nor in DNA methylation level of the analyzed region. Quantitative PCR showed no change in the the B4GALT1 gene transcriptional activity. The B4GALT1 gene has complex genetic and epigenetic regulation, and the effect of the RIOX1-dSpCas9 fusion protein could have gone unnoticed due to the narrow region in the gene promoter that was analyzed. Also, the already present epigenetic modifications and chromatin structure in the targeted region could have prevent the binding or catalytic activity of RIOX1-dSpCas9

Synthesis of fulleretic composite materials by liquid-directed mechanochemical templation

Predmet istraživanja je usmjeravajući učinak tekućine i fulerena u kontroliranoj mehanokemijskoj sintezi polimorfnih i kompozitnih faza cinkova imidazolata. Osim dosad poznatih faza cinkova imidazolata čije su strukture „preguste“ za apsorpciju fulerena, pripravljene su tri nove topologije kompozita cinkova imidazolata s fulerenom: C 70 @CHA- ZnIm 2 i C 60 @CHA-ZnIm 2 , C60 @X-ZnIm 2 i C 60 @T-ZnIm 2 . X i T faze su zasad nove i nepoznate topologije. Dobiveni spojevi su izučavani difrakcijom rentgenskih zraka na praškastim uzorcima, termogravimetrijom, infracrvenom spektroskopijom i elektronskom spinskom rezonancijom. Dok se CHA-ZnIm 2 i X-ZnIm 2 faza mogu pripraviti samo uz prisustvo fulerena, T-ZnIm 2 faza se može pripraviti i bez fulerena, ali manje količine enkapsuliranog fulerena dodatno stabiliziraju ovu nestabilnu fazu. Količina apsorbiranog fulerena ovisi o vrsti i količini dodanog fulerena (C 60 ili C70 ) i o tipu faze kompozita.The subject of research is the directing effect of liquid and fullerene in the controlled mechanochemical synthesis of polymorphic and composite phases of zinc imidazolate. In addition to the previously known zinc imidazolate phases whose structures are “too dense” for fullerene absorption, the inclusion of fullerene in the structure stabilized three new topologies of zinc imidazolate composites: C70 @CHA-ZnIm 2 and C 60 @CHA-ZnIm 2 , C 60 @X-ZnIm 2 and C 60 @T-ZnIm 2 . X and T phases have a new unreported topology. The obtained compounds were studied by X-ray diffraction on powder samples, thermogravimetry, infrared spectroscopy and electronic paramagnetic resonance. While the CHA-ZnIm 2 and X-ZnIm 2 phases can only be prepared with the addition of fullerene, T-ZnIm 2 phase can be prepared without the presence of fullerenes, but small amounts of encapsulated fullerene additionally stabilize the otherwise unstable phase. The amount of fullerene absorbed depends on the type of fullerene (C 60 or C 70 ), the amount of added fullerene and the type of composite phase

Development and validation of ultra-performance liquid chromatography method for the analysis of cinnarizine degradation products

Kemijska stabilnost aktivnih farmaceutskih sastojaka utječe na sigurnost i učinkovitost lijekova. U sklopu ovog diplomskog rada razvijena je, a zatim i validirana stabilitetno-indikativna metoda tekućinske kromatografije ultravisoke djelotvornosti uz detektor s diodnim nizom za praćenje kemijske stabilnosti cinarizina. Kako bi se razvila prikladna stabilitetno-indikativna analitička metoda najprije je provedena prisilna razgradnja na uzorcima cinarizina. Provedena je razgradnja pod utjecajem hidrolitičkih uvjeta (u neutralnom, kiselom i bazičnom mediju) pri temperaturi od 80 °C, razgradnja pod utjecajem oksidativnih uvjeta te fotolitička razgradnja cinarizina u otopini pri sobnoj temperaturi. Strukturna karakterizacija razgradnih produkata provedena je korištenjem spregnutog sustava tekućinska kromatografija ultravisoke djelotovornosti – spektrometrija masa visokog razlučivanja, uz eksperimente tandemne spektrometrije masa na ionima od interesa pri različitim vrijednostima kolizijskih potencijala.The chemical stability of active pharmaceutical ingredients affects the safety and effectiveness of drugs. As part of this thesis, a stability-indicative method of ultra-high efficiency liquid chromatography with a diode array detector for monitoring the chemical stability of cinnarizine was developed and then validated. To develop a suitable stability-indicative analytical method, forced degradation was performed on cinnarizine samples. Degradation under the influence of hydrolytic conditions (in neutral, acidic, and basic media) at a temperature of 80 °C, degradation under the influence of oxidative conditions and photolytic degradation of cinnarizine in solution at room temperature were carried out. Structural characterization of degradation products was carried out using a coupled system of ultra-high performance liquid chromatography – high resolution mass spectrometry, with tandem mass spectrometry experiments on ions of interest at different values of collision potentials

Nature based tourism in the slunjsko-plitvička area

Ovaj rad istražuje turizam temeljen na prirodi u slunjsko-plitvičkom kraju, s naglaskom na povezanost Nacionalnog parka Plitvička jezera s ostalim atrakcijama u blizini. Cilj istraživanja bio je analizirati motivaciju, aktivnosti, obilježja putovanja i zadovoljstvo posjetitelja, kao i ispitati turističku ponudu i infrastrukturu ovog područja. Podaci su prikupljeni putem anketnog istraživanja posjetitelja Nacionalnog parka i Rastoka. Rezultati istraživanja pokazuju visoko zadovoljstvo posjetitelja s prirodnim ljepotama i kulturnom baštinom, no također ukazuju na nedostatak koordinacije između različitih turističkih lokaliteta, što može rezultirati fragmentiranim iskustvom za posjetitelje. Iako postoje jasne prednosti u prirodnim resursima, infrastruktura nije dovoljno razvijena i usklađena s potrebama svih dionika, a povećan turistički pritisak stvara izazove u održavanju i očuvanju tih resursa. Istraživanje naglašava potrebu za integracijom svih ključnih atrakcija u jedinstvenu ponudu, koja bi omogućila lakšu povezanost između lokaliteta, te za održivim razvojem turizma koji poštuje prirodne i kulturne vrijednosti. Daljnji razvoj trebao bi uključivati bolju suradnju između lokalne zajednice, pružatelja usluga i turističkih dionika, čime bi se stvorila dugoročna održivost ovog područja.This thesis explores nature-based tourism in slunjsko-plitvička area, with a focus on the connection between Plitvice Lakes National Park and other nearby attractions. The aim of the research was to analyse visitor motivation, activities, travel characteristics, and satisfaction, as well as to examine the tourism offerings and infrastructure of the region. Data was collected through a survey conducted with visitors of Plitvice Lakes National Park and Rastoke. The findings show high visitor satisfaction with the natural beauty and cultural heritage, but also point to a lack of coordination between various tourist sites, which can result in a fragmented experience for visitors. While there are clear advantages in natural resources, the infrastructure is not sufficiently developed and aligned with the needs of all participants, and increased tourist pressure creates challenges in maintaining and preserving these resources. The research highlights the need to integrate all key attractions into a unified offering, which would improve connectivity between sites, and for sustainable tourism development that respects natural and cultural values. Further development should include better collaboration between the local community, service providers, and tourists, creating long-term sustainability for the area

Correlations and dynamics in many-body non-Hermitian systems

Ovaj rad istražuje međuigru temeljnih aspekata nehermitske kvantne mehanike i međudjelovanja čestica primjenom Bose-Hubbard modela s asimetričnim amplitudama preskoka i odbojnim interakcijama. U prvom dijelu analiziramo sustav s malim brojem bozona te pomoću računa smetnje i numeričkih metoda istražujemo razlike u odnosu na hermitski i jednočestični nehermitski slučaj. U nastavku proučavamo suprafluid-Mott izolator fazni prijelaz te ispitujemo kako nehermitska priroda sustava utječe na kritičnu vrijednost interakcije potrebnu za taj prijelaz.This thesis investigates the interplay between fundamental aspects of non-Hermitian quantum mechanics and particle interactions through the Bose-Hubbard model with asymmetric hopping and repulsive interaction. In the first part, we analyze a system with a small number of bosons and use perturbation theory and numerical methods to explore the differences compared to the Hermitian and single-particle non-Hermitian case. In the second part, we study the superfluid–Mott insulator phase transition and examine how system's non-Hermicity affects the critical value of interaction required for the transition to happen

DEVELOPMENT OF BOTANICAL CONCEPTS THROUGH ELEMENTARY AND SECONDARY EDUCATION

Ovim je istraživanjem analizirana usvojenost koncepata botanike kroz osnovnoškolsko i srednjoškolsko obrazovanje te međuodnosi sa stavovima i interesima učenika, studenata i nastavnika za botaniku. Istraživanje je diskontinuirano provođeno tijekom 15 godina, prije i poslije stupanja na snagu novog kurikuluma Prirode i Biologije te prije i poslije online nastave. Pismene provjere provedene su u tiskanom obliku ili online, a anketno istraživanje online. Utvrđeno je da je usvojenost botaničkih koncepata mala, a zbog neprimjerenih nastavnih programa dolazi do stvaranja brojnih miskoncepcija, od kojih neke nastaju i tijekom edukacije studenata nastavničkih profila. Interesi i stavovi učenika najveći su za procese u biljnom organizmu, a učitelja i nastavnika za ekologiju biljaka. Međutim, interesi i stavovi učenika ne mogu se povezati s usvojenošću koncepata botanike, kao ni uspješnost učenika s interesima nastavnika, koji smatraju važnim znanje o biljkama, ali dosadnom školsku botaniku. Na temelju ovog istraživanja identificirane su miskoncepcije i predložen potencijalni novi kurikulum botanike.This research analysed the acquisition of botany concepts through primary and secondary education and the interrelationships with the attitudes and interests of students and teachers in botany. The research was carried out discontinuously over 15 years, before and after the ratification of the new Nature and Biology curriculum and before and after online classes. Written tests were conducted in printed form or online, and polls only online. It has been established that the acquisition of botanical concepts is low, and due to inappropriate teaching programs, numerous misconceptions are created, some of which arise during the education of future teachers. Students' interests and attitudes are greatest for processes in the plant organism, and teachers for plant ecology. However, students' interests and attitudes cannot be linked to the acquisition of botany concepts, nor can students' success with the interests of teachers, who consider plant knowledge important but school botany boring. Based on this research, misconceptions were identified and a potential new botany curriculum was proposed