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Biological effects of novel n-methoxymethylated pyrimidine derivatives in human tumor cell lines
No full textU doktorskoj disertaciji istražili smo biološku aktivnost novih N-metoksimetiliranih (MOM) C-6 acikličkih derivata pirimidina, u uvjetima in vitro i in silico. Od svih spojeva, najsnažniji anti-proliferativni učinak na adherentne tumorske stanične linije je imao spoj 14b (6-(3-benziloksi-2-(benziloksimetil)propil-N,N-1,3-bis(metoksimetil)-5-metilpirimidin-2,4,dion), koji je ujedno pokazao i značajno jači citostatski učinak na leukemijskim stanicama K562 u usporedbi s klinički odobrenim lijekom Tasignom u uvjetima in vitro. In silico analiza pokazala je kako spoj 14b vjerojatno djeluje kao inhibitor RNA- i DNA- polimeraza, te kao inhibitor stanične adhezije. Daljnja in vitro istraživanja su potvrdila inhibitorni učinak spoja 14b na adheziju tumorskih stanica na podlozi sa fibronektinom, naročito kod metastatskih stanica kolorektalnog karcinoma SW620. Inhibitorni učinak spoja 14b na procese koje regulira DNA- polimeraza djelomično je bio potvrđen i analizom staničnog ciklusa koja je pokazala kako spoj 14b smanjuje postotak stanica SW620 u fazi staničnog ciklusa G1 te nakupljanje stanica u fazama staničnog ciklusa S i G2/M. Spoj 14b aktivira unutarnji (mitohondrijski) put apoptoze u stanicama SW620. Analiza staničnog metabolizma pokazala je i učinak spoja 14b na funkciju mitohondrija i glikolitičku aktivnost stanica u ovisnosti o koncentraciji, te je pri najnižoj koncentraciji od 0,1 μM uočena povećana metabolička aktivnost što upućuje na proliferativni učinak, dok je pri višoj koncentraciji od 1 μM, spoj 14b pokazao toksičan učinak na mitohondrije. Anti-proliferativna, pro-apoptotska i anti-adhezivna svojstva spoja 14b mogu se pripisati inhibiciji signalnih putova IGF-1R/FAK/Src, NF-κB/TGM2 i sfingozin kinaze 1, koji imaju važnu ulogu u kolorektalnoj karcinogenezi i metastatskim procesima. S obzirom na toksične učinke na normalnim fibroblastima čovjeka, potrebno je provesti daljnju optimizaciju i modifikaciju u svrhu dobivanja kandidata za daljnja predklinička istraživanja.In this doctoral thesis, the biological activity of novel pyrimidine derivates, N-metoxymethylated (MOM) C-6 acyclic pyrimidine derivates was evaluated in vitro and in silico. Out of all tested compounds, compound 14b (6-(3-benzyloxy-2-(benzyloxymetyl)propyl-N,N-1,3-bis(metoxymethyl)-5-methylpyrimidine-2,4,dion) showed the strongest antiproliferative effect on adherent tumour cell lines, as well as significantly stronger cytostatic effect in vitro on K562 leukaemia cell line in comparison to clinically approved drug Tasigna. In silico analysis showed that compound 14b likely works as an inhibitor of RNA and DNA polymerase, as well as an inhibitor of cell adhesion. Further in vitro research confirmed inhibitory effect of 14b compound on tumour cells adhesion on the fibronectin coated surface, especially in metastatic colorectal carcinoma SW620 cells line. Inhibitory effect of 14b compound on DNA polymerase regulated processes has also been partially confirmed by analysis of cell cycle showing 14b induced reduction of percentage of SW620 cells in the G1 phase of cell cycle, and accumulation of cells in S and G2/M cell cycle phases. Compound 14b activates intrinstic (mitochondrial) pathway of apoptosis in SW620 cells. Analysis of cell metabolism also showed concentration dependant effect of 14b on mitochondrial function and glycolitic activity. At lower concentrations of 0.1 μM increased metabolic activity was observed suggesting proliferative effect, while at the higer concentrations of 1 μM compound 14b showed the effect as mitochondrial toxicant. Antiproliferative, proapoptotic and antiadhezive effects of compound 14b may be attributed to the inhibition of signal pathways IGF-1R/FAK/Src, NF-κB/TGM2, and sphingosine kinase, which all have significant role in colorectal carcinogenesis and metastatic processes. Given the toxicity on normal human fibroblasts, further optimisation and modification is needed to obtain a candidate for subsequent preclinical research
Design and synthesis of novel pyrene-based pseudopeptide fluorophores and fluorescent nanomaterials
No full textSamoorganizirane peptidne nanostrukture su obećavajući biomaterijal s velikim potencijalom u
različitim biomedicinskim primjenama. Fluorescentno označavanje i oslikavanje stanica važna je
metoda za praćenje različitih bioaktivnih molekula u živim sustavima, zahvaljujući izuzetnim
prednostima, kao što su npr. jednostavna uporaba, visoka osjetljivost i visoka vremensko-prostorna
rezolucija.
U sklopu ove doktorske disertacije sintetiziran je niz fluorescentnih mono- (9a–d) i bis-pirenskih
derivata (4a–c), a njihova fotofizička svojstva i svojstva samoagregacije u otopini istražena su
UV/Vis i fluorescentnom spektroskopijom te dinamičkim raspršenjem svjetlosti (DLS). Piren je
kromofor s izvrsnim fluorescentnim kvantnim prinosom i učinkovitom emisijom ekscimera što ga
čini korisnim fluorescentnim senzorom. U polarnim otapalima (DMSO te smjesi otapala
DMSO/voda) mono-pirenski derivati (9a–d) pokazaju karakteristike monomerne emisije, dok bispirenski derivati (4a–c) pokazuju emisiju monomera i ekscimera, pri čemu se isti može pripisati
dinamičkom procesu. Proučavano je i ponašanje geliranja ovih spojeva te je samo za leucinski
mono-pirenski derivat 9a otkriveno da pokazuje geliranje u aromatskim otapalima kao što su toluen
i p-ksilen i u polarnoj smjesi otapala, voda/DMSO (4:1 v/v). Mono-pirenski derivati (9a–d) ne
pokazuju citotoksični učinak na stanične linije A549 i RD. Skoro svi derivati, osim derivata 4b,
pokazali su nakupljanje unutar živih i fiksiranih stanica, a makropinocitoza je utvrđena kao glavni
put za ulazak derivata 4a u stanice.Self-assembled peptide nanostructures are very promising biomaterials with huge potential in
several biomedical applications. Fluorescence imaging is emerging as promising and powerful
means for monitoring various bioactive molecules in living systems, which is thanks to its
remarkable advantages, such as easy operation, high sensitivity and high temporal-spatial
resolution.
As part of this doctoral dissertation series of fluorescent mono- (9a–d) and bis-pyrene derivatives
(4a–c) were synthesized and their photophysical and self-aggregation properties in solution, were
fully investigated by UV–vis and fluorescence spectroscopy and dynamic light scattering (DLS).
Pyrene is a chromophore with excellent fluorescent quantum yield and efficient excimer emission
that makes it useful as a fluorescence sensor. While in dilute DMSO mono-pyrene derivatives 9a–
d show features characteristic of monomer emission, bis-pyrene 4a–c display both monomer and
excimer emission, the latter attributable to a dynamic process. The gelation behaviour of these
compounds was also studied, and only mono-pyrene leucine derivative 9a was found to display
efficient gelation in aromatic solvents, such as toluene and p-xylene, and in polar solvent mixture,
water/DMSO (4:1 v/v). Finally, mono-pyrene 9a–d showed no apparent cytotoxic impact on A549
and RD cell lines; almost all derivatives, except derivative 4b, showed accumulation within both
live and fixed cells, and macropinocytosis was found to be the main pathway for derivative 4a to
enter into the cells
Effect of tripyridylporphyrin on melanoma cells
No full textMelanom je maligni tumor melanocita te je zbog svoje agresivnosti i metastatske prirode ujedno i najsmrtonosniji oblik raka kože. Liječenje melanoma uključuje kirurški zahvat, kemoterapiju, radioterapiju, biološku terapiju te lijekove koji specifično ciljaju mutacije povezane s melanomom. Nedostatak ovakvih oblika liječenja je nemogućnost nadilaženja rezistencije melanoma te su iz tog razloga današnja istraživanja usmjerena prema korištenju fotodinamičke terapije kao sigurne i učinkovite terapije. Fotodinamička terapija temelji se na reakciji svjetlosti određene valne duljine, lijeka osjetljivog na svjetlost (fotosenzibilizatora) i molekule kisika, pri čemu nastaju reaktivni kisikovi spojevi koji djeluju citotoksično. Brojnim istraživanjima dokazana je djelotvornost fotodinamičke terapije u liječenju melanoma, međutim još uvijek se provode istraživanja kako bi se pronašao najučinkovitiji fotosenzibilizator. Najčešće se proučavaju ciklički tetrapiroli, kao što su porfirini ispitani u ovom radu. U radu su korištena četiri amfifilna metilirana porfirina sa različitim duljinama lanca, 10C-M, 14C-M, 18C-M i AMIDO-MET. Cilj ovog istraživanja je odrediti djelovanje navedenih spojeva na stanice melanoma (MEWO) i fibroblasta (HFF) te utvrditi koji spoj pokazuje najbolji učinak. MTT testom dokazano je da spoj 18C-M u najvećoj mjeri smanjuje proliferaciju stanica, dok spoj AMIDO-MET ne utječe na proliferaciju. Fluorescentnim mikroskopom praćena je lokalizacija porfirina u stanicama te je utvrđeno da spojevi 10C-M, 14C-M i 18C-M ulaze u stanicu i nakupljaju se u citoplazmi, a prema intenzitetu crvenog obojenja utvrđeno je da se najviše nakuplja 18C-M. Zbog najveće duljine lanca, a samim time i najveće lipofilnosti, dokazano je da je spoj 18C-M najučinkovitiji fotosenzibilizator. Također, dokazano je da su spojevi podjednako učinkoviti u uvjetima sa i bez hipoksije te da nisu toksični u mraku.Melanoma is a malignant tumor of melanocytes and due to its aggressive as well as metastatic nature it is the deadliest form of skin cancer. Treatment of melanoma includes surgery, chemotherapy, radiotherapy, biological therapy, and drugs, which specifically target mutations associated with melanoma. The disadvantage of these therapies is inability to overcome resistance of melanoma and, because of that, studies are focused on using photodynamic therapy as a safe and effective therapy. Photodynamic therapy uses light of a certain wavelength, light-sensitive drug (photosensitizer) and oxygen molecule to form cytotoxic reactive oxygen species. Numerous studies have proven the effectiveness of photodynamic therapy in the treatment of melanoma; however, research is still being made to find the most effective photosensitizer. The most investigated photosensitizers are tricyclic tetrapyrroles, like porphyrins used in this study. Four methylated porphyrins with different chain lengths, 10C-M, 14C-M, 18C-M i AMIDO-MET, were used in this study. The aim of the study is to determine the effect of these compounds on melanoma cells (MEWO) and fibroblasts (HFF) as well as to determine which compound shows the best effect. The MTT assay showed that 18C-M had the biggest effect on reducing cell proliferation, while AMIDO-MET had no effect on proliferation. The localization of porphyrins in cells was monitored by fluorescence microscope and it was found that compounds 10C-M, 14C-M and 18C-M entered the cells and accumulated in cytoplasm. According to the intensity of red staining, it was found that 18C-M accumulates the most. Due to the longest side chain and the highest lipophilicity, it is proven that 18C-M is the most effective photosensitizer. Also, it is proven that all components are equally effective in conditions under and without hypoxia and they did not show dark toxicity
Uloge epigenetskih modifikacija u embrionalnoj i adultnoj neurogenezi
No full textThe entire nervous system develops from common neural stem cells (NSCs) that line the neural tube during the first weeks of embryonic development. NSCs are self-renewing, multipotent cells that generate neural progenitor cells by asymmetric division, and which, through a limited number of divisions, differentiate into specialized cells of the nervous system: neurons, oligodendrocytes, and astrocytes. Although most neurons are generated before birth, it has been shown that under the influence of appropriate stimuli, in adult mammals and humans, neurogenesis continues throughout life in limited regions of the brain; the subgranular zone of the dentate gyrus in the hippocampus and the subventricular zone of the lateral ventricles. Generation of neurons from NSCs encompasses successive stages of progenitor expansion, differentiation, maturation, and integration of new-born neurons into functional neural circuits. Numerous intrinsic and extrinsic factors are involved in the regulation of neurogenesis, forming a complex network of signalling pathways. It seems that epigenetic modifications and their cooperation play an integral role in the precise control of gene expression, which is crucial for the proper acquisition of neural fate. This review will comprehensively summarize the hitherto known roles of epigenetic modifications, in particular DNA modifications, histone modifications and non-coding RNA molecules, in regulating both embryonic and adult neurogenesis in the vertebrate CNS, as well as their mutual mechanisms of cooperation. Further elucidation of the role of epigenetic mechanisms in the process of neurogenesis could lead to the development of novel therapies needed for the treatment of neurodevelopmental and neurodegenerative disorders.Čitav živčani sustav razvija se iz zajedničkih neuralnih matičnih stanica (eng. neural stem cells, NSCs) koje oblažu neuralnu cijev tijekom prvih tjedana embrionalnog razvoja. NSCs su samoobnavljajuće, multipotentne stanice koje asimetričnom diobom generiraju neuralne progenitorske stanice, a koje kroz ograničen broj dioba diferenciraju u specijalizirane stanice živčanog sustava: neurone, oligodendrocite i astrocite. Iako većina neurona nastaje prije rođenja, dokazano je da se pod utjecajem odgovarajućih podražaja, u odraslih sisavaca i čovjeka, neurogeneza nastavlja tijekom cijeloga života u ograničenim regijama mozga; subgranularnoj zoni dentatnog gyrus-a u hipokampusu i subventrikularnoj zoni bočnih ventrikula. Stvaranje neurona iz NSCs uključuje uzastopne faze ekspanzije progenitora, diferencijaciju, sazrijevanje i integraciju novih neurona u funkcionalne neuralne krugove. U regulaciji neurogeneze sudjeluju brojni unutarnji i vanjski čimbenici, tvoreći kompleksnu mrežu signalnih puteva. Čini se da upravo epigenetske modifikacije te njihova međusobna suradnja igra sastavnu ulogu u preciznoj kontroli genske ekspresije ključnoj za pravilno usmjeravanje NSCs u stjecanje neuralne sudbine. Ovaj pregledni rad sveobuhvatno će sažeti do sad poznate uloge epigenetskih modifikacija, točnije DNA modifikacija, histonskih modifikacija i nekodirajućih RNA molekula, u regulaciji embrionalne i odrasle neurogeneze u centralnom živčanom sustavu kralježnjaka, kao i njihove međusobne mehanizme suradnje. Daljnje rasvjetljivanje uloga epigenetskih mehanizama u procesu neurogeneze mogao bi dovesti do razvoja novih terapija za liječenje neurorazvojnih i neurodegenerativnih poremećaja
The role of period genes in malignancies
No full textCirkadijalni ritmovi su fundamentalna osobina života, oni reguliraju biološke
procese koji se odvijaju tijekom 24 sata. Fiziološke i psihološke promjene
te promjene u ponašanju koje su usklađene s izmjenom dana i noći prate
taj 24-satni ciklus. Cirkadijalni sustav uključuje ulazni put koji odašilje
signale iz okoline do jezgre oscilatora (SCN) koja generira signale koji su
odgovorni za periferne fiziološke ili bihevioralne događaje.
Cirkadijalni ritmovi su samoodrživi, a prilagođavaju se signalima iz okoliša
uključujući svjetlost, temperaturu i hranu. Poremećaj u cirkadijalnim
ritmovima povezan je s nekoliko oblika raka u ljudima. Sve veća disfunkcija
rada sata već se pripisuje patogenezi raka, a mnoga su istraživanja otkrila
da je deregulacija cirkadijalnih satnih gena uključena u razvoj mnogih
bolesti, uključujući zloćudne bolesti. Nekoliko studija otkrilo je da su
smanjene razine ekspresije Period gena primijećene u tumorskim tkivima u
usporedbi s pripadajućim tumorom nezahvaćenim tkivima kod različitih
vrsta raka. Period geni su uključeni u molekularni mehanizam cirkadijalnog
sata te pokazuju svojstva tumor supresora, stoga su zanimljivi za
proučavanje. Deregulacija ovih gena predviđa smanjeno preživljenje u
bolesnika s uznapredovalim karcinomom pluća, bubrega, dojke i debelog
crijeva.Circadian rhythms are fundamental feature of life, they regulate biological
processes that take place over 24 hours. The circadian system includes an
input pathway that perceives signals from the environment and transmits
the input signals to the core oscillator (SCN) that generates signals that are
responsible for peripheral physiological or behavioral events.
Circadian rhythms are self-sustained and adapt to signals from the
environment including light, temperature, and food. Disturbances in
circadian rhythms have been associated with several forms of cancer in
humans. The increasing dysfunction of the clock is already attributed to the
pathogenesis of cancer, and many studies have found that deregulation of
circadian clock genes is involved in the development of various diseases,
including malignancies. Several studies have found reduced levels of Period
genes in tumor tissues compared to adjacent nontumorous tissues in
different types of cancer. Period genes are involved in the molecular
mechanism of circadian clock and show properties of tumor suppressor
genes, making them interesting to study. Deregulation of these genes
estimates reduced survival rates in patients with advanced lung, kidney,
breast, and colon cancers
Protein aggregation of DISC1 in mental illness
No full textDISC1 (poremećen u shizofreniji) je protein čiji je lokus gena povezan s mnogim mentalnim bolestima kao što su shizofrenija, depresija i bipolarni poremećaj. DISC1 protein ima mnogo uloga. Povezan je s neurogenezom, neurorazvojem, metabolizmom laktata, sintetičkom plastičnošću, dendritičkim rastom i unutarstaničnom trgovinom. Njegova mreža interakcija čini privlačnu metu za buduće terapeutske intervencije. U postmortalnim uzorcima mozga pacijenata koji imaju fenotipove afektivnih poremećaja ili shizofrenije, znanstvenici su otkrili frakcije DISC1 koje su identificirane kao netopivi proteinski agregati. Agregacija proteina biološki je fenomen koji se očituje kada se pogrešno složeni proteini nakupe i skupljaju unutar ili izvan stanice. Kako bi dublje istražili patologiju DISC1 proteina, znanstvenici su razvili transgeni model štakora koji prekomjerno izražava ne-mutirani ljudski DISC1 pune dužine u središnjem živčanom sustavu. Štakor tgDISC1 ima ulogu ispitivanja učinaka patologije DISC1 proteina, za razliku od nekih prethodnih životinjskih modela koji su imali ulogu testiranja učinaka mutacije DISC1 proteina. Eksperimenti na tgDISC1 štakorima pokazali su da: 1) DISC1 može djelovati kao senzor citosolnog oksidativnog stresa i ako reguliramo njegov sklop, to dovodi do nekih poremećaja u dopaminskoj homeostazi, 2) pogrešno sklapanje DISC1 može dovesti do različitih nepovezanih morfoloških fenotipa i 3) agregacija nemutiranog proteina može dovesti do poremećaja funkcije neuronskog kruga hipokampusa. Agregacija proteina DISC1 vrlo je važna za buduće eksperimente. Dobro je poznato da DISC1 protein može koagregirati s drugim proteinima koji su povezani mentalnim i neurodegenerativnim bolestima, pa bi daljnja istraživanja na tom polju bila od velikog interesa za bolji tretman pacijenata i također pri detekciji međusobnih poveznica između proteina koji vode do razvitka takvih bolesti.DISC1 (Disrupted in Schizophrenia) is a protein whose genetic locus is connected to many mental illnesses like schizophrenia, major depressive disorder, and bipolar disorder. DISC1 protein has many roles and is connected to neurogenesis, neurodevelopment, lactate metabolism, synaptic plasticity, dendritic growth, and intracellular trafficking. Its interaction network makes it an interesting target for future therapeutic research. In postmortem brain samples of patients that have schizophrenia or affective disorder phenotypes, scientists detected fractions of DISC1 that are identified as insoluble protein aggregates. Protein aggregation is a biological phenomenon that manifests when misfolded proteins accumulate and clump together either inside or outside of the cell. In order to investigate the DISC1 protein pathology in more detail, scientists developed a transgenic rat model which overexpresses full-length non-mutant human DISC1 in the whole body, including central nervous system. While previous animal models have looked at mutation or knockout of DISC1, this tgDISC1 rat allows us to examine the specific effect of DISC1 protein pathology. Experiments on tgDISC1 rat showed that:1) DISC1 can act as a sensor of oxidative stress in the cytosol and if we regulate its assembly, that leads to some disorder in dopamine homeostasis, 2) misassembly of DISC1 can lead to variety of unrelated morphological phenotypes and 3) aggregation of a nonmutant protein can lead to exacerbation in hippocampal neural circuit function. DISC1 protein aggregation is very important for future experiments. It is well known that DISC1 protein can co-aggregate with other proteins connected to mental illnesses and neurodegenerative diseases, so further research in that field can be of great interest for better treatment of patients and it could also help with detecting mutual links between proteins that can lead to development of such diseases
Glycomic and genetic biomarkers of posttraumatic stress disorder
No full textPosttraumatski stresni poremećaj (PTSP) je psihijatrijski poremećaj s kompleksnom etiologijom i bez validiranih biomarkera. Glikozilacija proteina je proces kovalentnog vezanja sećera na proteine te može značajno utjecati na njihovu biološku funkciju. Iako ovaj proces nema predefinirani kalup, postoji velik broj gena koji kodiraju za enzime i proteine uključene
u metabolizam i vezanje glikana. Cilj ovog istraživanja bio je analizirati glikozilacijski profil proteina u plazmi i imunoglobulina
G (IgG) kod ispitanika sa i bez PTSP-a te ispitati povezanost 12 polimorfizama s PTSP-om i zastupljenosti pojedinih N-glikana. N-glikom određen je pomoću visokoprotočne tekućinske kromatografije, a genotipizacija je provedena metodom lančane reakcije polimeraze u stvarnom vremenu. Razlike u N-glikomu između ispitanika sa i bez PTSP-a te između nositelja pojedinih genotipova i haplotipova odabranih polimorfizama, analizirani su u dvije neovisne kohorte. Ispitanici s PTSP-om imali su značajno promijenjen N-glikom plazme, ali ne i IgG-a, pri čemu su 4 N-glikana bila značajno povišena, a 2 značajno snižena u odnosu na kontrolne ispitanike. GG genotip s obzirom na HNF1A-AS1 rs7953249 bio je značajno rjeđe prisutan kod ispitanika
s PTSP-om, dok ostali testirani polimorfizmi nisu bili povezani s dijagnozom. 2 polimorfizma HNF1A su pokazali najveću povezanost s glikozilacijom u PTSP-u, odnosno sa sržno fukoziliranim IgG glikanima te visokorazgranatim N-glikanima u plazmi. 2 polimorfizma FUT8 bila su povezana sa sržno fukoziliranim N-glikanima, a B3GAT1 rs7928758 je bio značajno povezan s dva N-glikana u plazmi. Značajnost ostalih polimorfizama s glikozilacijom nije bila potvrđena u obje kohorte. Rezultati ove studije pokazali su sličnost N-glikoma u PTSP-u s onima primijećenima u upalnim procesima, autoimunim bolestima i drugim psihijatrijskim poremećajima te upućuju na potencijalnu regulatornu ulogu glikozilacije, upalnih čimbenika i HNF1A u razvoju i progresiji PTSP-a.Posttraumatic stress disorder (PTSD) is psychiatric disorder with complex etiology and no validated biomarkers. Protein glycosylation is a modification involving covalent binding of sugars on proteins and can radically change their biological properties. Although the process of sugar binding is not predefined, there are numerous genes that encode for the enzymes involved in metabolism and assembly of glycans. The aim of this study was to analyze plasma and immunoglobulin G (IgG) glycosylation patterns in subjects with and without PTSD and to examine the association of 12
polymorphisms with PTSD and distribution of specific N-glycans. N-glycome was determined using high performance liquid chromatography while genotyping was performed using realtime PCR. Differences in N-glycome between subjects with and without PTSD between carriers of different genotypes and haplotypes of selected polymorphisms, were anayzed in two separate cohorts. Patients with PTSD had significantly changed plasma, but not IgG, N-glycome, where 4 Nglycans were significantly higher and 2 significantly lower compared to control subjects. GG genotype carriers of HNF1A-AS1 rs7953249 were significantly less present in subjects with PTSD, while other polymorphisms were not associated with diagnosis. 2 polymorphisms HNF1A showed the highest association with glycosylation in PTSD, specifically with corefucosylated IgG N-glycans and highly branched and sialyated plasma glycans. 2 polymorphisms FUT8 were associated with core-fucosylated N-glycans, while B3GAT1 rs7928758 was associated with 2 N-glycans in plasma. Significant association of other polymorphisms with glycosylation was not replicated in both cohorts. This study showed similar glycosylation patterns in PTSD with ones seen in inflammatory processes, autoimmune diseases and other psychiatric disorders and implicates potential regulatory role of glycosylation, inflammatory factors and HNF1A in development and progression of PTSD
1,3-Dipolar cycloaddition reactions of tetra- and tripyridylporphyrins
No full text1,3-dipolarna cikloadicija jedna je od najvažnijih metoda u organskoj kemiji za sintezu peteročlanih prstena. Ovdje dolazi do reakcije između vrste zwitterionske rezonantne strukture, odnosno 1,3-dipola i spoja koji u svojoj strukturi ima višestruku vezu, odnosno dipolarofila. Jedna vrsta 1,3-dipola alilnog tipa su azometinski ilidi. Porfirini u 1,3-dipolarnoj cikloadiciji mogu sudjelovati kao dipolarofili i reagirati s azometinskim ilidima gdje nastaju klorini i bakterioklorini.
U ovom radu su Adler-Longo metodom sintetizirani 5,10,15,20-tetrakis(3-piridil)porfirin i 5-(4-acetamidofenil)-10,15,20-tris(3-piridil)porfirin te su dalje korišteni u reakcijama 1,3-dipolarne cikloadicije. Sinteze su provedene konvencionalnim putem i u mikrovalnim uvjetima. Oba porfirina su korištena za reakcije s dva različita azometinska ilida generirana in situ iz sarkozina i paraformaldehida te iz sarkozina i benzaldehida.
Reakcijom 1,3-dipolarne cikloadicije sintetiziran je novi spoj, monocikloadukt (klorin) 5,10,15,20-tetrakis(3-piridil)porfirina. Azometinski ilid je u ovoj reakciji generiran iz sarkozina i paraformaldehida, a reakcija je provedena konvencionalnim putem. Struktura monocikloadukta je potvrđena 1H i 13C NMR te UV/vis spektroskopijom. Apsorpcijski spektar novosintetiziranog monocikloadukta odgovara apsorpcijskom spektru karakterističnom za klorine. Novi monocikloadukt je okarakteriziran i fluorescencijskim spektrom. Produkti dobiveni u svim ostalim reakcijama provedenim konvencionalnim putem i u mikrovalnim uvjetima nisu izolirani kao čisti spojevi te struktura ovih spojeva nije mogla biti potvrđena NMR spektroskopijom.1,3-dipolar cycloaddition is one of the most important methods in organic chemistry for the synthesis of five-membered rings. This is the reaction between species represented by zwitterionic resonance structures (1,3-dipole) and multiple bond system (dipolarophile). Azomethine ylides are allyl type 1,3-dipoles. Porphyrins can participate as dipolarophiles in 1,3-dipolar cycloaddition with azomethine ylides; chlorins and bacteriochlorins can be obtained as a result of this reaction.
In this work, 5,10,15,20-tetrakis(3-pyridyl)porphyrin and 5-(4-acetamidophenyl)-10,15,20-tris(3-pyridyl)porphyrin are synthesized using Adler-Longo method and used in 1,3-dipolar cycloaddition reactions. Syntheses were carried out using conventional methods and microwave irradiation. Both porphyrins were used in 1,3-dipolar cycloaddition with two different azomethine ylides generated in situ. One was generated from sarcosine and paraformaldehyde and the other from sarcosine and benzaldehyde.
1,3-dipolar cycloaddition reaction of 5,10,15,20-tetrakis(3-pyridyl)porphyrin and azomethine ylide afforded monocycloadduct (chlorin). Azomethine ylide was in that case generated from sarcosine and paraformaldehyde. The reaction was carried out using conventional method. The structure of monoaduct was confirmed by 1H i 13C NMR and UV/vis spectroscopy. The absorption spectrum of monoaduct is typical of a chlorin. New monoaduct was also analyzed by fluorescence spectroscopy. Products obtained from other performed reactions, conventional and microwave assisted, were not isolated pure, therefore the structures of these compounds could not be confirmed by NMR spectroscopy
MOLECULAR EFFECTS OF NEW L-ASCORBIC ACID DERIVATIVES ON TRANSFORMED AND NORMAL HUMAN CELLS IN VITRO
No full textU ovom doktorskom radu istražen je biološki potencijal četiriju serija novih derivata L-askorbinske kiseline (L-ASA) (ukupno 67) koji se strukturno razlikuju s obzirom na supstituente na triazolnom prstenu i postojanje dvostruke veze između atoma C-4 i C-5. Ispitivanjem antiproliferacijskog učinka na tumorskim i netransformiranim staničnim linijama izdvojena su dva derivata (4e i 8m) sa selektivnim i najjačim učinkom na MCF-7 stanicama. Predviđeni mehanizam djelovanja svih derivata istražen je in silico metodom QSAR (eng. Quantitative structure – activity relationship) kojom je odabrana najvjerojatnija potencijalna meta, CDC25A (za 28,35% ispitanih derivata). PASS (eng. Prediction of Activity Spectra for Substances) – analizom pokazan je potencijalni kemoprotektivni učinak oba odabrana derivata. Biološki mehanizam djelovanja za derivate koji su pokazali značajan antiproliferacijski učinak dodatno je istražen i validiran analizom metaboličkog statusa i metodama proteomike. Signalni procesi regulirani faktorom hipoksije HIF-1α odabrani su kao najvjerojatniji potencijalni mehanizam selektivnog antiproliferacijskog učinka odabranih derivata. Antioksidativna aktivnost analizirana je DPPH metodom za one strukture s potencijalnim antioksidativnim svojstvima, te je ista bila izraţenija u derivata koji imaju 1,2,3-triazolil povezan s etilenskom poveznicom, s benziliranim laktonskim prstenom i C-6 supstituirani 1,2,3-triazolnim derivatima L-ASA s dvostrukom vezom u položaju C4=C5. Ispitivanjem staničnog metabolizma utvrĎeno je kako pojedini derivati iz grupe derivata sa slobodnim hidroksilnim skupinama na atomima C-2 i C-3 i iz grupe C-6 supstituiranih 1,2,3-triazolnih dibenzilnih derivata L-ASA nisu mijenjali funkciju mitohondrija. Zaključno, 1,2,3-triazolni derivati L-ASA nove su potencijalne protutumorske i antioksidativne strukture koje se daljnjom kemijskom optimizacijom mogu koristiti za razvoj agensa za protutumorsku terapiju ili kao antioksidanti.In this doctoral thesis, the biological potential of four series of new L-ascorbic acid (L-ASA) structurally different derivatives (67 in total) was investigated. The structures were different regarding the substituents on the triazole ring and double covalent bond between the C-4 and C-5 atoms. Based on the observed antiproliferative effect on tumor and untransformed cell lines, two derivatives (4e and 8m) with the most selective and strongest effect on MCF-7 cells were chosen for further investigation. The predicted mechanism of action of all derivatives was investigated in silico by the QSAR (Quantitative structure - activity relationship) method, which also proposed the most potential target protein of the selected derivates - CDC25A protein (determined for 28.35% of the derivatives tested). The results of PASS (Prediction of Activity Spectra for Substances) analysis showed the potential chemoprotective effect of both selected derivatives. The biological mechanism of action for derivatives that showed significant antiproliferative effect was further investigated and validated by metabolic status analysis accompanied by proteomics methods. The biological processes associated with hypoxia inducible factor - 1α (HIF-1α ) regulated cellular signaling have been proposed as potential mechanism of action of derivatives with the strongest antiproliferative effect. In addition, the structures that showed antioxidant properties were further subjected to the analysis of antioxidant activity by the DPPH method. The results of DPPH analysis revealed antioxidant activity for derivatives having a 1,2,3-triazolyl bonded with an ethylene linkage, with a benzylated lactone ring, and a C-6 substituted 1,2,3-triazole derivatives of L-ASA with double bond at position C4 = C5. In addition, the results of analysis of cellular metabolism showed that individual derivatives from the group of free hydroxyl groups at C-2 and C-3 atoms and from the group C-6 substituted 1,2,3-triazole dibenzyl derivatives of L-ASA did not affect mitochondrial function. In conclusion, 1,2,3-triazole derivatives of L-ASA are novel potential antitumor and antioxidant structures that can be used to develop anticancer agents or as antioxidants by further chemical optimization
Priručnik za praktikum opće kemije
No full textPriručnik čini 12 laboratorijskih vježbi koje se sastoje od teorijskog uvoda, ciljeva, popisa opreme, pribora i kemikalija potrebnih za izvođenje vježbe, postupka izvođenja te upute za prezentaciju rezultata u obliku skice, izračuna, grafičkog i tabličnog prikaza ili pisanog zaključka. Praktikum započinje pripremom otopina i metodama za razdvajanje pojedinih komponenti iz smjese: destilacijom, filtracijom, vakuum-filtracijom, ekstrakcijom, kromatografijom i kombiniranim metodama razdvajanja višekomponentne smjese, te se postupno stječu potrebne vještine za izvođenje narednih vježbi. Nakon što studenti steknu osnovna znanja i vještine za rad, u kemijskom laboratoriju izvode tematske vježbe: određuju standardni molarni volumen plinova kisika i vodika, entalpiju otapanja soli i reakcije neutralizacije, određuju provodnost elektrolita, sagledavaju kemijsku ravnotežu i Le Châtelierovo načelo kroz niz primjera kemijskih reakcija, upoznaju se sa zakonima kemijske kinetike kroz izračunavanje brzine kemijske reakcije, pripremaju acetatni i fosfatni puferski sustav i određuju kapacitet fosfatnog pufera. Praktikum završava elektrokemijskim procesima u sklopu kojih studenti sklapaju galvanske članke i mjere njihove elektrodne potencijale te provode postupak elektrolitičkog razlaganja vode