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Analiza strukturnih promjena DNA-DNA metiltransferaza 1 kompleksa tijekom vezivanja pomoću molekularne dinamike
No full textBackground: DNA methylation is the first and most enduring epigenetic mark, that acts as a fundamental mechanism in functional organization of human genome. Inhibitors and activators of DNA methylation can be used for genetic reprogramming of differentiated cells, or as tools for gene expression analysis. DNA substrates with guanine replaced by inosine at the target site showed an amazing 30 fold activation in methylation activity. Activation of mammalian DNA methyltransferase (Dnmt1) by inosine at the target base site was explored at the enzyme structure level in attempt to design activators of DNA methylation as novel drug-candidates.
Results: Coarse grained molecular dynamics simulations showed that mouse DNA methyltransferase forms unusual guanine-guanine base pair that can stabilize the catalytic complex. The stabilizing complex breaks down when the guanine at the target site is replaced by inosine. Principal component analysis with Bio3D application showed that the stability of the active site structure can affect Zn-finger rich domain that is distant from the active site.
Conclusion: Structural changes in Dnmt1-DNA complex showed that Zn-finger rich domains participate in stabilization of enzyme-DNA complex. The Zn-finger rich domain will be targeted in the future in silico drug design protocols in an attempt to design novel activators of DNA methylation in human cells.Pozadina:
DNA metilacija je prvi i najtrajniji epigenetički biljeg koji služi kao temeljni organizacijski mehanizam za ljudski genom. Inhibitori i aktivatori DNA metilacije mogu se koristiti za gensko reprogramiranje diferenciranih stanica ili kao alat za analizu genske ekspresije. Zamjena DNA supstrata s gvaninom, s DNA supstratom koji sadrži inozin na ciljnom mjestu pokazao je 30 puta veću aktivnost metilacije. Aktivacija DNA metiltransferaze 1 (Dnmt1) sisavaca od strane inozina na ciljnom mjestu istražena je na razini strukture enzima kako bi se potencijalno dizajnirali aktivatori DNA metilacije kao kandidati za nove lijekove.
Rezultati:
Simulacije molekularne dinamike koje koriste grubi model molekula pokazale su da mišja DNA metiltransfraza formira neobične gvanin-gvanin bazne parove koji mogu stabilizirati katalitički kompleks. Taj stabilizirajući kompleks se raspadne kada je gvanin zamjenjen s inozinom na ciljnom mjestu. Bio3D analiza komponenata pokazala je da stabilnost strukture aktivnog mjesta može utjecati na domenu bogatu “Zn-prst“ strukturama koja je udaljena od aktivnog mjesta.
Zaključak:
Strukturne promjene u DNA-Dnmt1 kompleksu pokazale su da domene bogate “Zn-prst“ strukturama sudjeluju u stabilizaciji enzim-DNA kompleksa. Domene bogate “Zn-prst“ strukturama predstavljaju potencijalne mete za buduće in silico protokole dizajna lijekova kako bi se dizajnirali novi aktivatori DNA metilacije u ljudskim stanicama
Adverse drug reactions of medicinal product belonging to ATC: D10 group reported to the Agency for Medicinal Products and Medical Devices
Get PDFAkne su upalno kožno oboljenje koje najviše pogađa osobe u adolescentskoj i pubertetskoj dobi, s mogućnošću nastavka bolesti u odrasloj dobi. Akne nastaju uslijed promjena u pilosebacealnoj jedinici. Iako su potrebna dodatna istraživanja o nastanku akni, mnogi ih znanstvenici dovode u vezu s genetičkom podlogom, prehranom, stresom, hormonskom neravnotežom te tipom kože pojedinca. Medicinsko liječenje akni ovisi o tipu akni. Ovaj rad donosi pregled svih nuspojava lijekova za liječenje akni (ATK D10) koje je Hrvatska agencija za lijekove i medicinske proizvode (HALMED) zaprimila u razdoblju od 1. siječnja 2018. do 31. prosinca 2018. godine. Analizirana je 141 prijava sumnji na nuspojave lijekova skupine D10, a rezultati su analizirani prema dobi i spolu pacijenta, prijavitelju, ozbiljnosti, ishodu, pripadnosti nuspojava prema klasifikaciji organskih sustava Medicinskog rječnika za regulatorne poslove (MedDRA) te djelatnoj tvari. Udio broja prijava nuspojava lijekova skupine D10 u ukupnom broju prijava nuspojava lijekova bio je 3,1%. Ukupno je analizirano 369 nuspojava lijekova od kojih se najveći broj odnosio na pacijente ženskog spola (64,5%) i dobnu skupinu od 18 do 44 godine (39,7%). Najviše nuspojava prijavljeno je od strane ljekarnika (43,3%) i nisu bile ozbiljne. Najčešće nuspojave lijekova skupine ATK D10 prijavljene su za azitromicin (23,1%), metilprednizolon (21,4%), klindamicin (13,3%), deksametazon (9,8%) te doksiciklin (8,7%), a najveći broj nuspojava rezultirao je oporavkom bez posljedica na pacijenta (35,2%). Podaci su uspoređeni s europskom i svjetskom bazom nuspojava lijekova te je vidljiva podudarnost u većini podataka.Acne is an inflammatory skin disease that mostly affects people in adolescence and puberty, with possibility of continuing the disease into adulthood. Acne is caused by changes in the pilosebaceal unit. Although more researches are needed on the onset of acne, many scientists have linked it to the genetic background, diet, stress, hormonal imbalance and skin type of an individual. The medical treatment for acne depends on the type of acne. This paper provides an overview of all side effects of medicinal product belonging to ATC: D10 group reported to the Croatian Agency for Medicinal Products and Medical Devices (HALMED) from January 1, 2018 to January 31, 2018. In total, 141 suspected adverse drug reactions from ATC:D10 group were analyzed and the results are divided by patient's age and gender, reporter qualification, seriousness criteria, outcome, affiliation of adverse reactions according to the Medical Dictionary of Regulatory Affairs (MedDRA) organic system classification and active substance. The proportion of reports of adverse drug reactions of D10 group in the total number of reports of adverse drug reactions was 3.1%. In total, 369 adverse drug reactions were analyzed and most of them are related to female patients (64.5%) and the 18-44 patient age group (39.7%). Most adverse reactions were reported by pharmacists (43.3%) and were not serious. The most common side effects for drugs from ATK: D10 group were reported for azithromycin (23.1%), methylprednisolone (21.4%), clindamycin (13.3%), dexamethasone (9.8%), and doxycycline (8.7%), and the highest number of side effects resulted in patient-free recovery (35.2%). The data are compared with the European and world-wide database of side effects of medicines, and there is a clear coincidence in most of the data
Antiproliferative effect of Eunicella Cavolini extracts and fractions on human foreskin fibroblasts
Get PDFGotovo 50% citotoksičnih spojeva izolirano je iz morskih organizama kao što su spužve i koralji. Koralji, uglavnom meki, potencijalni su izvori mnogih jedinstvenih metabolita, uključujući citotoksične i antitumorske spojeve. Ciljevi ovog istraživanja bili su analiziranje u kojem otapalu tijekom ekstrakcije dobijemo najveću masu ekstrakata te ispitivanje antiproliferativnog učinka dobivenih ekstrakata i frakcija na fibroblastima ljudskog prepucija. Pokazali smo kako je izbor polarnijeg otapala bolji pri ekstrakcijama Eunicelle cavolini. Nadalje, uzorak dobiven ekstrakcijom pomoću smjese otapala diklorometana i metanola frakcionirali smo na koloni te frakcije koristili za analiziranje antiproliferativnog učinka. Nakon MTT testova, najjači antiproliferativni učinak vidljiv je u djelovanju uzorka pripremljenog pomoću smjese heksana i etil-acetata (50:50) gdje je inhibicija proliferacije stanica za 50% vidljiva već pri koncentraciji od 0.69 µg/ml. Sličan, ali slabiji utjecaj na proliferaciju stanica imali su uzorci pripremljeni pomoću smjese etil-acetata i metanola (80:20) i čistog etil-acetata. Najmanja koncentracija potrebna za ubijanje 50% stanica također je dobivena kod uzorka pripremljenog pomoću smjese heksana i etil-acetata (50:50).Almost 50% of cytotoxic compounds are isolated from marine organisms such as sponges and corals. Corals, mainly soft corals, are potential sources of many unique metabolites including cytotoxic and anticancer compounds. The objectives of this study were to analyze the solvent extraction yield during the extraction and to investigate the antiproliferative effect of the obtained extracts and fractions on human foreskin fibroblasts. We have shown that the choice of polar solvents is better with the extracts of Eunicelle cavolini than the nonpolar solvent. Furthermore, the sample obtained by extraction with a mixture of dichloromethane and methanol solvent was fractionated on a column and these fractions were used to analyze the antiproliferative effect. After the MTT tests, the strongest antiproliferative effect is seen in the sample prepared by a mixture of hexane and ethyl acetate (50:50) where 50% cell proliferation inhibition is already apparent at a concentration of 0.69 μg/ml. Similar, but weaker, effect on cell proliferation had samples prepared using a mixture of ethyl acetate and methanol (80:20) and pure ethyl acetate. The minimum concentration required to kill 50% of the cells was also obtained in a sample prepared by a mixture of hexane and ethyl acetate (50:50)
Chiral brØnsted acid-catalyzed enentioselective synthesis of α-quaternary (triaryl)methanamines
No full textU sklopu ove disertacije pripravljena je serija α-(diaril) izoindolinonskih alkohola 1–24 koja je poslužila kao polazni supstrat za razvoj metodologije priprave kiralnih α-(3-indolil)(diaril)metanamina 26–60 i α-(triaril)metanamina 61–91.
Provedeno je detaljno ispitivanje priprave 3-supstituiranih 3-hidroksiizoindolinona upotrebom organometalnih reagensa. Fenilni supstituenti s elektron-donirajućim skupinama reagirali su s ftalimidom u Grignardovoj reakciji, dok su elektron-odvlačeće skupine i heteroaromatske jezgre zahtijevale litij-brom izmjenu ili strategiju izravne litijacije. Protokoli su tolerantni prema različitim funkcionalnim skupinama, a širok raspon 3-hidroksiizindolinona 1–24 dobiven je u dobrim i izvrsnim prinosima.
Razrađena je studija razvoja aza-Friedel-Craftsove asimetrične adicije indola na cikličke α-(diaril) supstituirane N-(acil)ketimine, generiranih in situ iz 3-aril-3-hidroksiizoindolinona. Enantioselektivna transformacija katalizirana kiralnim Brønstedovim kiselinama SPINOL-ne okosnice tolerira širok spektar različito supstituiranih indola i izoindolinonskih alkohola. Reakcijom su dobiveni α-kvaterni (3-indolil)(diaril)metanamini 26–60 u odličnim prinosima i razinama enantioselektivnosti (do 98 % prinosa, >99:1 e.r.). Izvor stereokemijske indukcije potkrijepljen je računalnim (DFT) i eksperimentalnim metodama.
Opisano je i istraživanje razvoja asimetrične Bettijeve reakcije fenola s α-(diaril) izoindolinonskim alkoholima 1–24 katalizirane kiralnim fosfornim kiselinama BINOL-ne strukture u svrhu dobivanja α-kvaternog (triaril) stereogenog centra. Reakcija je tolerantna na širok raspon supstituiranih 3-arilnih prstena izoindolinona 1–24, te na različite supstituente u para-položaju fenolnog nukleofila. Dobiveni α-(triaril)metanamini 61–91 izolirani su u dobrim do izvrsnim prinosima i umjerenim razinama enantioselektivnosti.Within this Thesis, a series of α-(diaryl) isoindolinone alcohols 1–24 were prepared which served as the starting materials for the development of a methodology for the preparation of chiral α-(3-indolyl)(diaryl) methanamines 26–60 and α-(triaryl) methanamines 61–91.
A detailed investigation of the synthesis of 3–substituted 3-hydroxyisoindolinones with organometallic reagents is described. Phenyl groups with electron-donating substituents are easily introduced by employing Grignard reaction, while aryls with electron-withdrawing groups and heteroaromatic cores require lithium exchange or direct lithiation strategy. The protocols are tolerant of various functional groups, and a wide range of 3-hydroxyisoindolinones 1–24 were afforded in good to excellent yields.
The study of asymmetric aza-Friedel-Crafts reaction between indoles and cyclic α-diaryl-substituted N-acyl imines, generated in situ from 3-aryl 3-hydroxyisoindolinones, is described. The enantioselective transformation proceeds smoothly with a broad range of indoles and isoindolinone alcohols using a SPINOL-derived chiral Brønsted acid catalyst to afford α-quaternary (3-indolyl)(diaryl)methanamines 26–60 in excellent yields and enantioselectivities (up to 98% yield, up to >99:1 e.r.). The origin of stereochemical induction is supported by DFT calculations and experimental data.
The study of asymmetric Betti reaction between phenol derivatives and α-(diaryl) isoindolinone alcohols 1–24 catalyzed by chiral phosphoric acids for the construction of α-quaternary (triaryl) stereogenic centre is developed. The reaction is tolerant to a wide range of 3-aryl substituents on 3-hydroxyisoisoindolinones 1–24, as well as in the para-position of the phenol nucleophiles. The resulting α-(triaryl) methanamines 61–91 were isolated in good yields and moderate levels of enantioselectivity
Mass Spectrometry of silver(I) Schiff base complexes
Get PDFReakcijom razrijeđene octene kiseline i para-nitrofenilhidrazina s para-metoksiacetofenonom, odnosno s para-nitrobenzaldehidom u molarnom omjeru 1 : 1 priređena su dva nova derivata hidrazina, C13H10N4O4 i C15H15N3O3. Pripravljeni spojevi identificirani su pomoću elementne analize, dok je njihova struktura u otopini okarakterizirana 1D i 2D tehnikama spektroskopije NMR (1H, 13C, 15N). Reakcijom srebrova(I) nitrata s pripravljenim ligandima istražen je utjecaj različitih otapala (DMSO-d6, CD3CN-d3, CDCl3-d) na vezanje liganda i metala u omjeru 1:1, [(AgL(NO3)]. Koncentracijskim titracijama praćenima snimanjem 1H NMR spektara uzoraka istražen je množinski omjer vezanja te mjesto koordinacije liganda na metal. Pripravljeni kompleksi okarakterizirani su 1H i 1H-15N HMBC tehnikama NMR te spektrometrijom masa (ESI–MS).Two new hydrazine derivatives, C13H10N4O4 and C15H15N3O3 were prepared by diluting acetic acid and para-nitrophenylhydrazine with para-methoxyacetophenone or para-nitrobenzaldehyde in a molar ratio of 1:1. The prepared compounds were identified by elemental analysis, while their structure in the solution was characterized by 1D and 2D NMR spectroscopy techniques (1H, 13C, 15N). Different solvents effects on ligand and metal binding at molar ratio 1:1 [(AgL(NO3)), were investigated with the reaction of silver(I) nitrate with prepared ligands (DMSO-d6, CD3CN-d3, CDCl3-d). Concentration titrations followed by the 1H NMR spectra of the samples investigated the multivalent binding ratio and the ligand coordination site on the metal. The prepared complexes were characterized by 1H and 1H-15N HMBC NMR techniques and mass spectrometry (ESI-MS)
CHRONOTHERAPY AND CIRCADIAN RHYTHM IN CANCER TREATMENT
No full textIstraživanje cirkadijalnog ritma ističe kako ritmovi održavaju zdravlje.
Cirkadijalni vremenski sustav odgovoran je za predviđanje promjena u
podnošljivosti i djelotvornosti antitumorskih lijekova te u zloćudnom širenju ili
rastu tumora. Satni geni cirkadijalnog vremenskog sustava povezani su u
regulatorne povratne petlje, a njihovo djelovanje usklađeno je
suprahijazmatskom jezgrom koja također prilagođava cirkadijalne ritmove
ciklusima okoliša i vanjskim podražajima. Cirkadijalni ritam ciklus je u trajanju
od približno dvadeset i četiri sata. Poremećaj cirkadijalnog ritma ima veliki
utjecaj na staničnu diobu i razvoj raka i obrnuto: maligna transformacija
uzrokuje poremećaje cirkadijalnog sata. Najpovoljnije vrijeme primjene lijeka
unutar dvadeset i četiri sata, tzv. kronoterapija, može dovesti do značajnog
napretka u sigurnosti terapije raka. Administracija lijeka u skladu s
cirkadijalnim vremenom može pomaknuti dosadašnju terapiju raka s principa
''veća toksičnost, bolja učinkovitost'' na ''bolja podnošljivost, veća
učinkovitost''. Zbog toga što se mnogi antitumorski lijekovi razlikuju u
potenciji i/ili toksičnosti te tako različito utječu na ritmičnost biokemijskih,
fizioloških i bihevioralnih procesa, cirkadijalni ritam može utjecati na
učinkovitost liječenja raka moduliranjem farmakokinetike i farmakodinamike
lijekova. Kako bi se primjena lijeka sinkronizirala s bolešću i cirkadijalnim
ritmovima, potrebni su prilagodljivi i primjereni sustavi isporuke terapije. Na
kraju, u ovome su radu proučena neka od istraživanja koja, kao cilj, imaju
individualizaciju liječenja raka uzimajući u obzir cirkadijalni ritam. Ovaj rad
ističe da kronoterapija može odigrati ključnu ulogu u kvaliteti života i stopi
preživljavanja onkoloških bolesnika.Circadian rhythm research points out that rhythm maintain health. The
circadian timing system is responsible for predicting changes in tolerability
and efficacy of antitumor agents, and in malignant promotion or tumor
growth. Circadian clock genes are linked to regulatory feedback loops, and
their function is aligned with a suprachiasmatic nucleus that also adjusts the
circadian rhythms to environmental cycles and external stimuli. The circadian
rhythm cycle lasts approximately twenty-four hours. Disturbance of circadian
rhythm has a major influence on cell division and cancer development, and
vice versa; malignant transformation causes disorders of the circadian clock.
Adequate time of administration of the drug within twenty-four hours, called
chronotherapy, can lead to significant progress in the safety of cancer therapy.
The administration of the drug in accordance with circadian time can move
the existing cancer therapy from the principle of "higher toxicity, better
efficacy" to "better tolerability, greater efficacy". Because many anticancer
drugs differ in potency and/or toxicity and thus affect differently the rhythm
of biochemical, physiological and behavioral processes, circadian rhythm can
affect the efficacy of cancer treatment by modulating drug pharmacokinetics
and pharmacodynamics. In order to synchronize drug administration with
disease and circadian rhythms, adaptive and appropriate drug delivery
systems are needed. Finally, some of the research aimed at individualizing
cancer treatment according to the circadian rhythm has been reviewed. This
final thesis shows that chronotherapy can play a key role in quality of life and survival rates for oncological patients
Liquid chromatography-mass spectrometry method for identification and quantification of sildenafil in dietary supplements for erectile dysfunction
Get PDFPojava krivotvorina rastući je problem na farmaceutskom tržištu. Među najkrivotvorenijima su lijekovi za erektilnu disfunkciju. Međutim, vrlo su česte i patvorine dodataka prehrani koje sadrže sintetske lijekove za erektilnu disfunkciju.
U ovom radu opisana je validacija metode tekućinske kromatografije spregnute sa masenom spektrometrijom za identifikaciju i određivanje sadržaja sildenafila u dodacima prehrani kod kojih se sumnja na krivotvorenje sintetskim lijekovima za erektilnu disfunkciju.
Za separaciju spojeva tekućinskom kromatografijom korištena je C18 kolona. Otopina koncentrirane formijatne kiseline u ultračistoj vodi i otopina koncentrirane formijatne kiseline u metanolu korištene su kao mobilna faza. Analiza je započeta identifikacijom sildenafila u uzorcima metodom masene spektrometrije. Za detekciju i kvantifikaciju sildenafila korišten je UV detektor pri valnoj duljini 280 nm, kao i kvadrupolni analizator i analizator vremena leta (Q-TOF). Metoda se pokazala prikladnom za analizu dodataka prehrani.
Korištenjem navedene metode sildenafil je detektiran u dva dodatka prehrani u prosječnoj koncentraciji većoj od 90,0 mg po dozi.Pharmaceutical counterfeiting is a growing problem. One of the most counterfeited class of drugs and dietary supplements are erectile dysfunction drugs and supplements.
This paper describes the validation of the liquid chromatography-mass spectrometry method for identification and quantification of sildenafil in dietary supplements for erectile dysfunction and analysis of dietary supplements which are considered to be adultered.
Liquid chromatography was performed on C18 stationary phase. A solution of formic acid in methanol and a solution of formic acid in ultrapure water was used as the mobile phase. Analysis began by identification of sildenafil using mass spectrometry. For detection and quantification of sildenafil, UV-detector (wavelength: 280 nm), as well as quadrupole and time of flight mass analysers were used. This method was shown to be suitable for analysis of dietary supplements.
Using this method sildenafil was identified and quantified in two dietary supplements in the concentration of 90,0 mg per dose
Optimisation of tetrahydrocannabinol and metabolites analysis by gas chromatography-mass spectrometry in rat urine
Get PDFZbog rastućeg javnog mišljenja o antitumorskom potencijalu tetrahidrokanabinola (THC) te kao sredstvu za ublažavanje simptoma kemoterapije irinotekanom, dio onkoloških bolesnika poseže za neregistriranim pripravcima THC-a. Da bi se procijenio utjecaj THC-a na metabolizam irinotekana na eksperimentalnom modelu štakora, potrebno je razviti osjetljivu, točnu i preciznu analitičku metodu kojom se može odrediti masena koncentracija THC-a te njegovih metabolita [11-hidroksi-delta-9-tetrahidrokanabinola (THC-OH) i 11-nor-delta-9-tetrahidrokanabinol-9-karboksilne kiseline (THC-COOH)] u urinu štakora kojima je apliciran samo THC, te u grupi kojoj je istovremeno apliciran THC i irinotekan. U tu su svrhu optimirani uvjeti hidrolize i ekstrakcije ispitivanih analita te se proveo razvoj i validacija plinsko-kromatografske metode uz detekciju spektrometrom masa (GC/MS) za istovremeno određivanje sva tri ispitivana analita. Optimalnom metodom hidrolize THC, THC-OH i THC-COOH glukuronida, pokazala se kombinirana hidroliza, enzimom β-glukuronidazom iz E. coli na 50 °C tijekom 2 sata, nakon koje je slijedila alkalna hidroliza. Ekstrakcija na čvrstoj fazi prilagođena za kanabinoide korištena je prije GC/MS analize. Predloženom metodom analiziran je istovremeno sadržaj sva tri analita u urinu štakora prikupljenog unutar 24 sata od tretmana (n = 10). THC nije detektiran niti u jednom uzorku, THC-OH je detektiran u 50 % uzoraka, a THC-COOH u svim uzorcima. Metoda opisana u radu zbog svoje je osjetljivosti (granice detekcije: 0,8-1 μg/L), točnosti (> 96 %) i preciznosti (RSD 96%) and precision (RSD < 6%)
Screening of proteins with the potential to aggregate in mental illness
No full textŠizofrenija, klinička depresija i bipolarni poremećaj spadaju u grupu kroničnih mentalnih bolesti zbog njihove cijeloživotne i ponavljajuće prirode. Kronične mentalne bolesti jedan su od najvećih uzroka nesposobnosti za rad u Europi i globalno. Postojeće metode liječenja uključuju prihoterapiju i lijekove poput antipsihotika i antidepresiva. Moderna istraživanja pokušavaju unaprijediti dijagnozu i liječenje istraživanjem bioloških mehanizama koji mogu biti povezani s bolestima. Rezultati novih istraživanja, umjesto na samo simptomološke, fokusirali bi dijagnozu i liječenje na biološke faktore. Jedan od napredujućih bioloških faktora koji se istražuje je agregacija proteina. Nedavna istraživanja otkrila su nekoliko proteina koji agregiraju u moždanom tkivu bolesnika. Ova otkrića impliciraju da bi mehanizam agregacije proteina mogao biti ključan za razvoj mentalnih bolesti. Kako bismo proširili ova istraživanja, u moždanim tkivima pacijenata identificirani su novi proteini. U ovom istraživanju ispitivano je 12 proteina koji su identificirani u pročišćenoj insolubilnoj proteinskoj frakciji moždanih tkiva bolesnika. Vrsta proteina i njihova količina u uzorku identificirani su masenom spektroskopijom. Ispitivani proteini potencijalni su kandidati za stvaranje agregata u mentalnim bolestima. Kako bismo mogli eksprimirati proteine bilo je potrebno gene premjestiti u ekspresijski vektor pomoću Gateway Sistema. Eksprimirani proteini analizirani su Western blot metodom i fluorescentnom mikroskopijom. Od 12 ispitivanih proteina, 6 je dokazano Western blot analizom i fluorescentnim mikroskopom. Od dokazanih proteina dva su od većeg značaja za ovo istraživanje: jedan agregira, dok drugi pokazuje jaku indikaciju za agregacijom. Daljnja ispitivanja za ova dva proteina su prioritet za buduća istraživanja.Schizophrenia, major depressive disorder and bipolar disorder can be referred to as chronic mental illnesses because of their lifelong and recurrent nature. They are amongst the most disabling diseases in Europe and globally. Existing methods of diagnosing and treating chronic mental illnesses are psychotherapy, as well as treatment with antipsychotics and antidepressants. Modern research projects concerning mental illnesses are attempting to improve these methods by trying to identify the biological pathways involved in these illnesses. These findings would focus treatment on the biological factors rather than only on symptoms. One rising factor being investigated is protein aggregation. Recent discoveries have found that a few proteins form aggregates in brain samples taken from patients suffering from chronic mental illnesses. These findings imply that protein aggregation could be one of the mechanisms contributing to the development of mental disorders. To expand these studies, novel proteins are being identified in brain samples from patients with mental illnesses. In this study, we investigated 12 proteins which were identified in the purified insoluble fractions from brain samples from the patients. The proteins and their quantity were identified with mass spectroscopy. These proteins may represent new aggregating proteins, and so we aimed to confirm if they have the ability to aggregate. To be able to express the genes in the mammalian cells they were transferred using the Gateway cloning system into the expression vector. When expressed they were analyzed by Western blot and fluorescent microscopy. Out of 12 investigated proteins, 6 were proven by Western blot and fluorescent microscopy, of which one was clearly seen to form aggregates, with the strong indication that another would as well. Further study of these two proteins should, therefore, be a priority for future research. Key words: mental illness, protein aggregation, novel protein
Characterization of bioactive compounds from grape juice and pomace by using mass spectrometry methods and examination of juice enrichment potential
No full textPolifenoli dokazano imaju vrlo jako antioksidativno, kardioprotektivno, neuroprotektivno i antikancerogeno djelovanje zbog čega je uvriježena nutricionistička preporuka za konzumacijom proizvoda bogatih polifenolima, što uključuje i grožđe te u umjerenim količinama i vino. Međutim, vino je proizvod koji nije namijenjen za konzumaciju u cijeloj populaciji te je u ovom diplomskom radu istražena i predstavljena alternativna opcija pića bogatog polifenolima koja bi bila pogodna i za skupine populacije poput starijih, djece i trudnica. Stoga je glavni cilj ovog istraživanja bio razvoj funkcionalnog proizvoda - soka od grožđa, obogaćenog polifenolima, kojeg mogu konzumirati sve skupine ljudi. Metodama masene spektrometrije i spektrofotometrijskim analizama je ispitan sastav polifenola soka, komine i obogaćenih sokova od grožđa. U ovom istraživanju su se koristili uzorci grožđa autohtonih bijelih sorti Kastavštine, za obogaćivanje sokova grožđa sa kominama, kako bi se bolje ispitao njihov, dosad neistraženi, polifenolni profil. Dobiveni rezultati ukazuju na to kako je međusobni utjecaj određenog soka i određene komine vrlo bitan, odnosno ovisno o porastu koncentracije željenog polifenola ključno je odabrati sortu komine za obogaćivanje soka od grožđa određene sorte. Time krajnji proizvod, obogaćeni sok ima specifičan polifenolni profil. Etanol, kao prihvatljivo otapalo u industrijskoj proizvodnji hrane, se pokazao učinkovit u ekstrakciji polifenola iz komine.
Obogaćeni sokovi imaju visoki eksploatacijski potencijal u industriji te predstavljaju visokokvalitetan proizvod, koji široj populaciji može biti dobar izvor bioaktivnih spojeva. Također bi se ciljanim povezivanjem proizvodnje funkcionalnih sokova mogla pripremiti projekcija smanjenja organskog otpada i povećanja ekonomske isplativosti iskorištenja grožđa. Naposljetku bi proizvodnja autohtonih funkcionalnih proizvoda uvelike doprinijela regionalnom i nacionalnom gospodarskom rastu i razvitku.Polyphenols have very strong antioxidative, cardioprotective, neuroprotective and anticancerogenic activity, so accordingly, it is nutritionally recommended to increase consumption of polyphenol-rich food, such as for example grape products and moderately wine. Unfortunately, wine is a product that is not intended for the consumption of the entire population, and, within the presented research alternative polyphenol-rich beverage from grape has been studied and presented that might be appropriate for consumption in population groups of elderly, pregnant women and children. That is why the main goal of this research was development of a functional product – grape juice enriched with polyphenols, which is appropriate for consumption within all population groups. By use of mass spectrometry and spectrophotometry based methods, composition of polyphenols in juice and pomace as well as in enriched juices was examined. In this research samples of autochthonous white grapes from Kastav were exploited, to analyse their, until now, unexplored polyphenol profile, useful for future usage in enrichment of grape juice products. Results show that mutual influence of specific juice and specific pomace is highly relevant, in other words, rise in concentration of desired polyphenol is the key to the choice of specific sort if pomace for enrichment of the grape juice of specific sort. In such a way, a specific polyphenol profile for enriched product may be achieved. Furthermore, ethanol, as an appropriate solvent in industrial production of food, has shown to be efficient in extraction of polyphenols from the pomace.
Enriched juices have great potential in industrial exploatation as they are a high quality end product adequate as a good source of bioactive compounds to the wider population. In addition, such targeted industrial development might be linked with development of projections for reduction of organic waste and increase in economic profitability. At last, production of autochthonous functional products might significantly contribute to the regional and national economic growth and development