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UTJECAJ ISTOVREMENE MANIPULACIJE DNA I HISTONA NA EKSPRESIJU GENA UKLJUČENIH U EPITELNO-MEZENHIMSKU TRANZICIJU PRIMJENOM SUSTAVA CRISPR/dCas9
No full textInterplay between epigenetic modifications is essential for regulation of gene expression.
The adaptation of CRISPR/Cas9 technology for epigenetic research by introducing dead Cas9
(dCas9) fused to epigenetic effector domains (ED) has enabled significant advancements in this
field. I expanded the CRISPR/dCas9 modular system with five histone modifying domains
(G9a, G9a-me3, LSD1, KDM5A, macro1.1) and DNMT3L domain, as well as the multi-guide
module to accommodate up to 11 gRNAs. From a set of candidate genes involved in epithelial
to mesenchymal transition (EMT), ZEB1 and SNAI1 were selected for further epigenetic
manipulations in HepG2 cell line. Their CpG islands were simultaneously targeted with dCas-
DNMT3A combined with specific dCas9-ED fusions to understand the interplay of DNA
methylation and histone modifications. The combinations had synergistic effect on gene
expression and affected the EMT phenotype. Interplay between introduced modifications was
also observed as a locus specific synergistic increase in DNA methylation, and was not
disrupted by global histone acetylation increase after sodium butyrate treatment. The
combinatorial epigenetic effects were not replicated in A549 cell line, suggesting specificity of
epigenetic regulatory pathways.Međudjelovanje epigenetičkih modifikacija je neophodno regulaciju ekspresije gena.
Korištenje CRISPR/Cas9 tehnologije u epigenetičkim istraživanjima pomoću inaktivnog Cas9
(dCas9) fuzioniranog s epigenetičkim efektorskim domenama (ED) omogućilo je značajan
napredak u ovom području. Proširila sam modularni sustav CRISPR/dCas9 s pet domena za
modifikaciju histona (G9a, G9a-me3, LSD1, KDM5A, makro1.1) i domenom DNMT3L, te
„multi-guide“ modul koji može primiti do 11 gRNA. Od nekoliko kandidat gena koji sudjeluju
u epitelno-mezenhimskoj tranziciji (EMT), odabrala sam ZEB1 i SNAI1 za daljnje epigenetičke
manipulacije u staničnoj liniji HepG2. Njihovi CpG otoci su istovremeno ciljani s DNMT3AdCas9
u kombinaciji s određenom dCas9-ED fuzijom kako bi se istražilo među-djelovanje
DNA metilacije i modifikacija histona. Kombinacije su imale sinergistički učinak na ekspresiju
gena i utjecale su na EMT fenotip. Simultano uvedene modifikacije pokazale su sinergistički
efekt na razini metilacije DNA na određenom lokusu, koji nije bio osjetljiv na povećanje
globalne acetilacije histona. Kombinacije dCas9 fuzija nisu djelovale na ekspresiju ciljanih
gena u A549 staničnoj liniji, što ukazuje na specifičnost epigenetičkih regulatornih putova
Učinci povišene temperature vode i onečišćujućih tvari na nekoliko bioloških obilježja ličinki tulara
No full textUrbanization and inadequate wastewater treatment contaminate freshwater ecosystems with
complex chemicals, which, along with climate change, threaten freshwater insects. To assess
these impacts, we conducted a 21-day microcosm experiment simulating a simplified
freshwater food web containing moss and larvae of two caddisfly species, Drusus croaticus
and Allogamus uncatus. The experiment included control (C), increased temperature (T2 = C +
4 °C), emerging contaminants mixture (EC), and multiple stressor treatment (MS = EC + T2).
We analysed caddisfly mortality, body weight, locomotor activity (distance moved), and
compared species-specific effect of selected stressors. ECs exposure increased average body
weight in both species with contrasting effects on locomotor activity, increasing larval average
distance moved in D. croaticus but reducing it in A. uncatus. T2 treatment caused the highest
mortality for D. croaticus larvae, while the mortality of A. uncatus larvae was similar in all
treatments. MS treatment significantly impacted D. croaticus larvae behaviour, leading to
reduced larval average distance moved compared to other treatments, highlighting the
complexity of multiple stressor interactions. The observed interspecies variability emphasizes
the importance of considering species-specific characteristics in ecological risk assessments.
These findings offer insights into how multiple stressors may impact caddisfly populations,
crucial members of aquatic ecosystems.Urbanizacija i neadekvatna obrada otpadnih voda onečišćuju slatkovodne ekosustave smjesama
onečišćivača, koji s klimatskim promjenama ugrožavaju slatkovodne kukce. U svrhu procjene
tih utjecaja, proveli smo 21-dnevni eksperiment stimulirajući pojednostavljenu slatkovodnu
hranidbenu mrežu s mahovinom i ličinkama dviju vrsta tulara, Drusus croaticus i Allogamus
uncatus. Eksperiment je uključivao kontrolnu skupinu (C), tretman s povišenom temperaturom
(T2 = C + 4 °C), smjesom onečišćivača (EC) te višestrukim stresorima (MS = EC + T2).
Analizirali smo smrtnost, srednju tjelesnu težinu, lokomotornu aktivnost (srednja prijeđena
udaljenost) tulara te usporedili učinak odabranih stresora specifično na svaku vrstu. Izloženost
smjesi onečišćivača povećala je prosječnu tjelesnu težinu u obje vrste, s kontrastnim učincima
na lokomotornu aktivnost, povećavajući prosječnu prijeđenu udaljenost ličinki D. croaticus te
je smanjujući za ličinke A. uncatus. Povišena temperatura (T2) uzrokovala je najveću smrtnost
ličinki D. croaticus, dok je smrtnost ličinki A. uncatus bila slična u svim tretmanima. Tretman
višestrukim stresorima značajno je utjecao na ponašanje ličinki D. croaticus, smanjujući
prosječnu prijeđenu udaljenost u usporedbi s kontrolnom skupinom i drugim tretmanima,
naglašavajući složenost interakcija višestrukih stresora. Uočena varijabilnost među vrstama
naglašava važnost razmatranja karakteristika specifičnih za pojedinu vrstu. Naša otkrića
pružaju uvid u utjecaj višestrukih stresora na populacije tulara, ključne članove vodenih
ekosustava
Role of inhibitory receptors LAG-3, TIM-3 and PD-1 in the pathogenesis of hemorrhagic fever with renal syndrome
No full textInhibicijski receptori nadziru aktivaciju i diferencijaciju stanica tijekom prepoznavanja ciljnih
antigena slanjem koinhibicijskih signala koji reguliraju jačinu imunosnog odgovora. Hemoragijska
vrućica s bubrežnim sindromom (HVBS) bolest je koju uzrokuju ortohantavirusi Starog svijeta, a
najčešći uzročnik u Europi je Puumala ortohantavirus. Poznato je da imunosni odgovor ima ulogu
u patogenezi HVBS-a. Cilj istraživanja bio je ispitati povezanost razina ekspresije receptora LAG-
3, TIM-3 i PD-1 na limfocitima CD8+ i prirodnoubilačkim stanicama i njihove koncentracije u
serumu s kliničkim parametrima koji ukazuju na težinu kliničke slike u pacijenata oboljelih od
HVBS-a. Metodama imunotest za istovremenu detekciju različitih proteina Procartaplex i protočna
citometrija izmjerene su razine ekspresije receptora LAG-3, TIM-3 i PD-1 na limfocitima CD8+ i
prirodnoubilačkim stanicama i njihove koncentracije u serumu te je ispitana njihova povezanost s
kliničkim parametrima koji ukazuju na težinu kliničke slike. Rezultati su pokazali veću razinu
ekspresije ovih receptora na limfocitima CD8+ i prirodnoubilačkim stanicama i veću koncentraciju
njihovih solubilnih oblika u serumu pacijenata oboljelih od HVBS-a u odnosu na kontrole te
njihovu povezanost s kliničkim parametrima. Dodatna istraživanja potrebna su kako bi se utvrdila
uzročno-posljedična veza između razina ekspresije receptora LAG-3, TIM-3 i PD-1 i težine
kliničke slike te dodatno ispitala njihova uloga u akutnim infekcijama.Inhibitory receptors control the activation and differentiation of cells during the recognition of
target antigens by sending co-inhibitory signals that regulate the strength of the immune response.
Hemorrhagic fever with renal syndrome (HFRS) is a disease caused by Old World
orthohantaviruses, and the most common causative agent in Europe is Puumala orthohantavirus. It
is known that the immune response plays a role in the pathogenesis of HFRS. The aim of the study
was to explore the association of LAG-3, TIM-3 and PD-1 receptor expression on CD8+
lymphocytes and natural killer cells and their serum concentrations with clinical parameters that
indicate the severity of the clinical picture in patients with HFRS. Procartaplex multiplex
immunoassay and flow cytometry were used to measure the expression of LAG-3, TIM-3 and PD-
1 receptors on CD8+ lymphocytes and natural killer cells and their concentrations in serum, and
their association with clinical parameters indicating the severity of the clinical picture was
examined. The results showed a higher expression of these receptors on CD8+ lymphocytes and
natural killer cells and higher concentrations of their soluble forms in the serum of patients with
HFRS compared to controls, and their association with clinical parameters. Additional research is
needed to determine the causal relationship between the expression of LAG-3, TIM-3 and PD-1
receptors and the severity of the clinical picture and to further examine their role in acute infections
Symmetric functionalization of Anderson-type polyoxomolybdates
No full textIstražene su mogućnosti funkcionalizacije Andersonovog heteropolioksomolibdata s organskim i metaoloorganskim spojevima. Sintetiziran je simetrično funkconalizirani spoj [N(C4 H9 )4] 3[CoMo6 O18{(OCH2 )3CNH2 }2], a potom njegovom reakcijom s hidrazonskim kompleksom bakra [Cu3 (μ 3 -NO3){(C5 H4N)CH=N–N(C=O)(C5 H4 N)}3 (H2 O)3 ]n(NO3 )2n·3nH2O dobiven je hibrid složene strukture. Koordinacijom aminskih skupina funkcionaliziranog polioksometalata na atome bakra, polianion povezuje dva periferna koordinacijska polimera u jedinstvenu strukturu. Analognom reakcijom s [Cu(NO3 ){(C5 H4 N)2CH=N–NH(C=O)(C5 H4 N)}]2 (NO3 )2 nije bilo moguće prirediti hibrid sličnog sastava. Sljedeći pristup temeljen na reakciji [Cu2(OAc)4 (3-pyCHO)2] s [N(C4 H9 )4]3[CoMo6 O18{(OCH2 )3CNH2 }2] nije doveo do povezivanja dvaju podjedinica već do nastajanja imina. Direktnom reakcijom [N(C4 H9 )4] 3[CoMo6 O18{(OCH2 )3CNH2 }2] s 4-piridinkarbaldehidom priređen je heteropolioksometalat [N(C4 H9 )4]3[CoMo6 O18{(OCH2 )3C=NCHC5 H4 N}2].The functionalization of Anderson-type heteropolyoxomolybdate with organic and metalorganic compounds was investigated. Symmetrically functionalized compound [N(C4 H9 )4] 3[CoMo6 O18{(OCH2 )3CNH2 }2] was prepared, and afterwards, by its reaction with copper hydrazone [Cu3 (μ 3 -NO3){(C5 H4N)CH=N–N(C=O)(C5 H4 N)}3 (H2 O)3 ]n(NO3 )2n·3nH2 O complex, a hybrid with a complex structure was obtained. The polyanion connects the two peripheral coordination polymers into a unique structure by coordinating the amine groups of the functionalized polyoxometalate to the copper atoms. By analogous reaction with [Cu(NO3 ){(C5 H4 N)2CH=N–NH(C=O)(C5 H4 N)}] 2 (NO3 )2, it was not possible to prepare a hybrid with a similar composition. The next approach was based on the reaction of [Cu2(OAc)4 (3-pyCHO)2] with [N(C4 H9 )4] 3[CoMo6 O18{(OCH2 )3CNH2 }2], but this method did not result with the interconnection of the two subunits, but instead the imine formed. The heteropolyoxometalate [N(C4 H9 )4]3[CoMo6 O18{(OCH2 )3C=NCHC5 H4 N}2] was prepared by direct reaction of [N(C4 H9 )4] 3[CoMo6 O18{(OCH2 )3CNH2 }2] with 4-pyridinecarbaldehyde
Phytochemical composition and antioxidant capacity of aqueous extracts of broccoli (Brassica oleracea L. convar. botrytis var. cymosa) seedlings grown at high temperature
No full textKlimatske promjene i povremeni temperaturni ekstremi utječu na razvoj biljaka za opskrbu prehrambene i ostalih industrija koje koriste biljni materijal kao sirovinu. Kako bi preživjele, biljke svoj kemijski sastav prilagođavaju okolišnim uvjetima, što može uzrokovati i promjenu u nutritivnoj vrijednosti. Svojte iz porodice kupusnjača (Brassicaceae) vrlo su zastupljene u prehrani, a sve veću popularnost stječe i tzv. “mikropovrće“ tj. klijanci zbog kratkog vremena uzgoja, manje potrebnog prostora i resursa, te visoke nutritivne vrijednosti. Brokula pripada najzastupljenijim kupusnjačama u ljudskoj prehrani, čija bi nutritivna vrijednost mogla biti ugrožena zbog porasta okolišne temperature. Stoga je cilj ovog diplomskog rada bio istražiti utjecaj simuliranih uvjeta visoke temperature okoliša na udio primarnih i specijaliziranih metabolita, te antioksidacijski kapacitet vodenih ekstrakata klijanaca brokule (Brassica oleracea L. convar. botrytis (L.) Alef. var. cymosa Duch.). Spektrofotometrijski je određen udio ukupnih fenola, flavonoida, flavonola, hidroksicimetnih kiselina, antocijana, glukozinolata, proteina i topivih šećera. Metodom tekućinske kromatografije visoke moći razlučivanja razdvojeni su, identificirani i kvantificirani L-askorbinska, sinapinska i ferulična kiselina, te flavonoidi kempferol i kvercetin. Visoka temperatura uzrokovala je povećanje udjela ukupnih fenola, flavonola i šećera, kao i koncentracije ferulične i sinapinske kiseline, kvercetina i kempferola. S druge strane, udio ukupnih flavonoida i L-askorbinske kiseline je smanjen. Metodom ABTS zabilježeno je povećanje antioksidacijskog potencijala, a metodom DPPH smanjenje.Global warming has become one of the main challenges of modern civilizations. Climate changes and temperature extremes, affect plant production for food and other industries that use them. Plants can adapt to environment by changing phytochemical composition, which could have a significant impact on plant nutritional value. Species from Brassicaceae family are intesely represented in diet and there is also an increase in usage of „microgreens“ because of shorter growth period, resource usage and good nutritional value. As one of main Brassicaceae species present in the human diet, broccoli production could become endagered because of higher temperature environment condition, so the aim of this work was to investigate metabolic response and the impact of high temperature conditions on primary and secondary metabolites, and antioxidant capacity water extract of broccoli seedlings (Brassica oleracea L. convar. botrytis (L.) Alef. var. cymosa Duch.). The content of total phenols, flavonoids, hydroxycinnamic acids, flavonols, soluble sugars and proteins was determined spectrophotometrically. Using a high-performance-liquid-chromatography method, ferulic, sinapic and L-ascorbic acid, kaempferol and quercetin, were separated, identified and quantified. The antioxidant capacity was determined by methods ABTS, DPPH and FRAP. Higher temperature increased ferulic and sinapic acid, quercetin and kaempferol concentration, total phenols, flavonols and sugars. ABTS method showed and increase in antioxidant capacity, whereas the DPPH showed a decrease. The research also showed a decrease in flavonoids and L-ascorbic acid. The ABTS method showed an increase in the antioxidant potential, while DPPH showed a decrease
Neuroprotective activity of cholinesterase reactivators in organophosphate poisoning
No full textKationski piridinijevi oksimi koriste se kao reaktivatori inhibiranih enzima acetilkolinesteraze (AChE) i butirilkolinesteraze (BChE) pri liječenju trovanja organofosfornim (OP) živčanim bojnim otrovima i pesticidima. Međutim, neučinkoviti su za reaktivaciju sinaptičke AChE u mozgu zbog otežanog prolaska kationa kroz krvno-moždanu barijeru, što može potaknuti neuroupalu i trajno oštetiti moždane funkcije. Cilj ovog istraživanja bio je ispitivanje neuroprotektivnog potencijala triju novosintetiziranih skupina oksima s obzirom na poboljšane fizikalno-kemijske karakteristike u odnosu na piridinijeve oksime. Od osam oksima kationske imidazolijeve skupine, 1,3-dibenzil-2-((hidroksiimino)metil)imidazolijev bromid pokazao se učinkovitim reaktivatorom fosfilirane BChE s kojom je kao pseudokatalitički par u ex vivo uvjetima unutar 5 minuta vratio 60 % enzimske aktivnosti, što ga čini izvrsnim kandidatom za razgradnju OP spojeva u cirkulaciji prije nego dođu do ciljnih tkiva. Također, tri zwitterionska acetamidna bis-oksima na modelu miša pokazala su bolji zaštitni indeks i manju akutnu toksičnost od standardnih oksima. Terapija jednim od bis-oksima vratila je oko 70 % aktivnosti inhibirane AChE u mozgu i značajno je smanjila ekspresiju neuroupalnih biljega astroglioze i mikroglioze. Analizirani bis-oksimi imaju kapacitet sprječavanja aktivacije neuroupalnih procesa i pokazuju perspektivna fizikalno-kemijska svojstva za razvoj poboljšane terapije u slučaju trovanja OP spojevima.Cationic pyridinium oximes are used as reactivators of inhibited acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in the treatment of poisoning with organophosphorus (OP) nerve agents and pesticides. However, they are ineffective in reactivating synaptic AChE in the brain due to the difficulty of cation passage through the blood-brain barrier, which can induce neuroinflammation and permanently damage brain functions. The goal of this research was determining the neuroprotective potential of three newly synthesized oxime groups with regard to improved physicochemical characteristics compared to pyridinium oximes. Out of the eight cationic imidazolium oximes,
1,3-dibenzyl-2-((hydroxyimino)methyl)imidazolium bromide proved to be an effective ex vivo reactivator of phosphorylated BChE and as a pseudocatalytic pair it restored 60 % of the enzyme activity with a 10-fold excess cyclosarin, which makes it an excellent candidate for the degradation of OP compounds in the circulation before they reach the target tissues. Three zwitterionic acetamide bis-oximes showed a better protective index and less acute toxicity than standard oximes in a mouse model. Therapy with one of the bis- oximes restored about 70 % of the inhibited AChE in the brain and significantly reduced the expression of neuroinflammatory markers of astrogliosis and microgliosis. The analyzed bis- oximes have the capacity to prevent the activation of neuroinflammation and show promising physicochemical properties for the improvement of therapy in case of OP poisoning
One-pot synthesis of a fully protected trisaccharide from linear oligosaccharide domain of Quillaja saponaria plant saponin
No full textU sklopu ovog diplomskog rada sintetiziran je potpuno zaštićeni trisaharid β-D-Xylp-(1→4)-α-L-Rhap-(1→2)-D-Fucp koji je ujedno i strukturna domena saponina QS-21 izoliranog iz biljke Quillaja saponaria, nužna za njegovu imunoadjuvantsku aktivnost. Metoda sinteze glikozidne veze korištena u ovom diplomskom radu koristi glikozil-fluoride kao donore glikozila te sililne etere kao akceptore. Za razliku od standardnih reakcija glikozilacije, ova metoda ne zahtijeva rigorozne suhe uvjete, niti hlađenje na niske temperature, već se odvija uz komercijalno dostupan katalizator, borov trifluorid dietileterat, pri sobnoj temperaturi i u otapalu koje ne mora biti sušeno. Korištenje navedenih prekursora otvara mogućnost tzv. one-pot glikozilacije, odnosno povezivanja nekoliko reakcija glikozilacije u jedan sintetski korak. Strategija one-pot glikozilacije korištena u sintezi ciljnog trisaharida iskorištava suptilne razlike u reaktivnosti različitih sililnih etera. Konačni prekursori i produkt sinteze strukturno su okarakterizirani standardnim spektroskopskim metodama (IR, 1H i 13C NMR).n this diploma thesis, a fully protected trisaccharide β-D-Xylp-(1→4)-α-L-Rhap-(1→2)-D-Fucp was synthesized. This trisaccharide is a structural domain of saponin QS-21, which is isolated from the plant Quillaja saponaria and is essential for its immunoadjuvant activity. The glycosidic bond synthesis method used in this thesis employs glycosyl fluorides as glycosyl donors and silyl ethers as acceptors. Unlike standard glycosylation reactions, this method does not require rigorous dry conditions nor cooling to low temperatures. Instead, it utilizes a commercially available catalyst, boron trifluoride diethyl etherate, at room temperature and in a solvent that does not need to be dried. The use of these precursors opens up the possibility of one-pot glycosylation, connecting several glycosylation reactions into a single synthetic step. The one-pot glycosylation strategy used in this synthesis exploits subtle differences in the reactivity of various silyl ethers. The final precursors and the product were structurally characterized using standard spectroscopic methods (IR, 1H, 13C NMR)
Sadržaj metala u eluatima tla s područja bivše tvornice glinice kod Obrovca i njihov učinak na klijanje sjemenki salate (Lactuca sativa)
No full textAlumina factories are plants for refining aluminum oxide from bauxite ore. Seven soil samples were taken in the vicinity of the former Jadral alumina factory near Obrovac, and the multielement analyses revealed elevated concentrations of heavy metals, especially iron (Fe), but high levels of Mn, Cr, V and Zn were also detected. Two types of soil eluates were prepared from the soil samples using two methods - the EN 12457-2 (European Standard Batch Leaching Test) and the TCLP-2 (Toxicity Characteristic Leaching Procedure). The soil eluates differed mainly in the pH values, while the metal content was generally low. The seed germination test was carried out not only with soil eluates prepared according to the two methods mentioned, but also with eluates whose pH was adjusted to 6.5. In addition to the influence of multielement composition of the eluates, the pH value had a predominant effect on both the germination of the lettuce seeds and the radicle growth. Comparing the effects of the samples on radicle growth and the germination process, radicle growth was the more sensitive parameter. This study sheds light on the impact of the former alumina factory, by showing the presence of heavy metals in the soil and possible consequences for the environment.Tvornice glinice su postrojenja za rafiniranje aluminijevog oksida iz boksitne rude. U sklopu ovog istraživanja uzeto je sedam uzoraka tla u blizini bivše tvornice Jadral kod Obrovca. Multielementna analiza otkrila je povišene koncentracije teških metala u tim uzorcima, posebice željeza (Fe), kao i visoke razine Mn, Cr, V i Zn. Kako bi se detaljnije istražili ovi nalazi, dvije vrste eluata tla pripremljene su iz uzoraka tla korištenjem dviju različitih metoda - EN 12457-2 (European Standard Batch Leaching Test) i TCLP-2 (Toxicity Characteristic Leaching Procedure). Dobiveni eluati tla razlikovali su se prvenstveno u pH vrijednosti, dok je njihov sadržaj metala općenito bio nizak. Uz navedeno, istražen je utjecaj eluata na klijavost sjemenki salate, ne samo eluata tla pripremljenih prema gore navedenim metodama, već i eluata čija je pH vrijednost podešena na 6,5. Rezultati su pokazali da je uz multielementni sastav uzoraka eluata tla, značajan bio utjecaj pH vrijednosti, kako na klijavost sjemenki salate tako i na rast korijenka. Uspoređujući učinke uzoraka na rast korijenka i proces klijanja, rast korijenka bio je osjetljiviji parametar. Ovo istraživanje ukazuje na utjecaj bivše tvornice aluminija, ističući prisutnost teških metala u tlu i moguće posljedice za okoliš
Development and validation of ultra-performance liquid chromatography method for the analysis of cinnarizine degradation products
No full textKemijska 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
Višestruko istovremeno dokiranje malih molekula u aktivno mjesto butirilkolinesteraze primjenom dubokog pojačanog učenja
No full textCholinesterase inhibitors are often used in treating Alzheimer’s disease. Molecular docking
represents an important tool in the design of novel inhibitors. In this study, a multicomponent
quantum chemical Monte Carlo semi flexible molecular docking of four selected components of
the Ugi reaction was performed inside the active site of butyrylcholinesterase (BChE). The IC 50
value of compounds was evaluated, and artificial neural networks (ANNs) were trained by deep
reinforcement learning for two of them, using results from docking. Even though components
alone do not inhibit BChE significantly, formaldehyde and benzylamine can be used jointly for
the design of novel drugs because they bind closely to the enzyme’s catalytic triad. In addition,
the advantage of using the three component Ugi reaction is highlighted. Finally, ANN was shown
to be successful in representing a potential energy surface of docking simulations. O btained ANN
can be used as a scoring function for future dockings, with significantly shorter calculation time.U liječenju Alzheimerove bolesti često se koriste inhibitori kolinesteraza. Molekularno dokiranje važan je alat u dizajnu novih inhibitora, a u ovome je radu unutar aktivnog mjesta butirilkolinesteraze (BChE) provedeno multikomponentno kvantno-kemijsko Monte Carlo polufleksibilno dokiranje četiriju komponenata Ugijeve reakcije. Određene su im i vrijednosti IC50 te su za dvije od njih istrenirane umjetne neuronske mreže (ANN) primjenom dubokog pojačanog učenja za opis rezultata dokiranja. Iako zasebne molekule ne inhibiraju značajno BChE, formaldehid i benzilamin zajedno mogu poslužiti u dizajnu novih lijekova jer se vežu u blizini katalitičke trijade enzima. Uz to, istaknuta je prednost uporabe trokomponentne Ugijeve reakcije. Naposlijetku, pokazano je da se ANN može uspješno koristiti u reprezentaciji plohe potencijalne energije dokiranja. Dobivena mreža može ubuduće poslužiti kao funkcija bodovanja, uz znatno kraće trajanje proračuna