Antitimocitni globulini su poliklona protutijela koja dovode do smanjenja broja T stanica. Često se koriste za imunosupresiju primatelja tijekom transplantacije bubrega. Timoglobulin su IgG ...protutijela dobivena imunizacijom zečeva timocitima ljudskog fetusa. Timoglobulin dovodi do smanjenja broja T-limfocita putem razgradnje stanica mehanizmom ovisnim o komplementu te apoptozom. Da bismo dokazali direktan učinak timoglobulina na stanice, koristili smo ljudske embrionalne stanice porijeklom iz bubrega (HEK-293) u staničnoj kulturi. Mjerili smo membranski potencijal stanice metodom prikovanih potencijala. Depolarizacijski učinak timoglobulina na membranski potencijal HEK-293 stanica je ovisan o koncentraciji timoglobulina te o početnom membranskom potencijalu stanica. Učinak timoglobulina u hipoksično-reoksigenacijskih uvjetima utvrđen je određivanjem stanične smrtnosti te stanične migracije testom grebanja. Timoglobulin je smanjio staničnu smrtnost u hipoksijskim uvjetima nakon kojih je slijedila reoksigenacija. Ovo je prvo istraživanje koje pokazuje direktan učinak timoglobulina na HEK-293 stanice. Rezultati ovog istraživanja potiču nova istraživanja o djelovanju timoglobulina na stanice transplantiranih bubrega.
Antithymocyte globulines are polyclonal T cell-depleting immunoglobulins used in induction of immunosuppression in kidney transplant recipients. Thymoglobuline, is purified rabbit IgG, obtained by immunization of rabbits with fetal human thymi, which depletes T lymphocytes by complement-dependent lysis and apoptosis. To determine possible direct effects of Thymoglobuline on the cells, we used Human Embryonic Kidney cell line (HEK-293) in culture. We measured membrane potential of the cells using slow whole patch clamp technique. Depolarizations of HEK-293 cells caused by Thymoglobuline were concentration- and membrane potential- dependent, showing direct effect of Thymoglobuline on the HEK-293 cells. The effects of Thymoglobuline in hypoxia/reoxigenation were detected by calculating cell death and determining the cell migration using scratch test. Thymoglobuline prevented the cell death induced by hypoxia and reoxigenation conditions. This is first study showing direct effect of thymoglobulin on HEK-293 cells. The results of this study encourage the research on epithelial cells in transplanted kidneys.
Prekomjerno stvaranje reaktivnih kisikovih spojeva (ROS) u stanicama uzrokuje ireverzibilne modifikacije proteina, kao što je na primjer karbonilacija, koje utječu na njihovu strukturu i funkciju te ...na stabilnost proteoma. Istraživanja provedena na bakterijama i beskralješnjacima su pokazala postojanje korelacije između oštećenja proteoma i stanične smrti. Cilj ovog rada bio je utvrditi postoji li takva poveznica između stanične smrti i oštećenja proteina imortalnih staničnih linija dobivenih iz primarnog tumora (SW480) i metastaze (SW620) adenokarcinoma debelog crijeva. Izlaganjem staničnih linija oksidativnom stresu utvrdili smo da stanična linija SW620 ima nižu razinu oštećenih proteina od stanica SW480 te je otpornija na oštećenja proteoma izazvanih UVC zračenjem. Razlog povećane otpornosti proteoma stanica SW620 mogla bi biti niska razina ROS-a u odnosu na stanice SW480 i veća stabilnost proteoma. Usporednom analizom proteoma staničnih linija SW480 i SW620 u trenutku kada je smrtnost u populaciji iznosila 90% utvrdili smo da su mogući razlozi povećane osjetljivosti stanica SW480 narušene funkcije sustava za održavanje proteostaze i odstranjivanja oštećenih proteina te smo identificirali proteine koji bi mogli biti potencijalne mete u razvoju protutumorskih terapija.
The excessive production of reactive oxygen species (ROS) in cells induces irreversible protein modifications, carbonylation, that impairs protein structure and function, as well as proteome stability. The correlation between proteome damage and cell death is well established in bacteria and invertebrates and the aim of this research was to investigate the relationship between mortality and protein damage of immortal cell lines derived from primary tumor adenocarcinoma SW480 and its metastasis SW620. We found that SW620 cell line is more resistant to UVC-induced oxidative damage relative to SW480 cells. The reason for greater resistance of SW620 cells could be lower ROS level compared to SW480 and greater proteome stability. The differential analysis of proteomes of two cell lines after 90% UVC-induced mortality shows that SW480 displays greater sensitivity due to the damage of proteome quality system and the ability to remove oxidative damaged proteins. Also, we identified proteins that could be possible targets in anti-cancer therapy.
Uvod i cilj
Nedostatak S-adenozilhomocistein hidrolaze (AHCY) je poremećaj uzrokovan mutacijama u genu ahcy čime je smanjena aktivnost proteina AHCY. AHCY ima ključnu ulogu u pravilnom odvijanju ...ciklusa aminokiseline metionina u stanici, stoga nedostatak njegove funkcije uzrokuje težak metabolički poremećaj. Kao jedini enzim koji hidrolizira SAH, snažni inhibitor staničnih metiltrasferaza, AHCY ima i indirektnu ulogu u održavanju metilacijskog statusa stanice. Klinička slika ovog potencijalno letalnog oboljenja je karakterizirana kombinacijom mišićnih, neuroloških i jetrenih poremećaja. Unatoč dokazanoj esencijalnosti AHCY, promjene u molekularnim mehanizmima stanice u stanju nedostatka AHCY su dosada nedovoljno istražene. Cilj ove doktorske disertacije je analiza i razumijevanje molekularnih i staničnih uloga AHCY s krajnjom svrhom probira potecijalnih biomarkera oboljenja.
Materijali i metode
U sklopu ove disertacije su rađene studije novootkrivene Y328D mutante potvrđene u pacijentici oboljeloj od nedostaka funkcije AHCY, a za dodatna istraživanja na raspolaganju su bili i primarni fibroblasti pacijentice. Zbog ograničenja u radu s primarnim fibroblastima, tijekom izrade doktorske disertacije su se pripremili, validirali i istraživali modelni stanični sustavi za što se koristila kombinacija metoda klasične biologije i različitih omiks i visokoprotočnih tehnika.
Rezultati
Po prvi puta je tijekom istraživanja AHCY korištena metoda kojom se na temelju fluorescencije dviju komplementiranih podjedinica proteina Venus s kojima su označeni proteini od interesa može pratiti njihova interakcija u ljudskim stanicama. Dokazano je da Y328D mutanta gubi sposobnost homodimerizacije, a metoda je također uspješno prilagođena na visokoprotočno skeniranje interaktora proteina AHCY. Nadalje, uočena je smanjena razina mutiranih AHCY mRNA i proteina u fibroblastima pacijenta i u stanicama sa stabilno eksprimiranim mutiranim AHCY. U istim stanicama je također mikroskopijom pokazano da mutacije uzrokuju povećanu ili smanjenu količinu proteina u jezgri stanica u odnosu na divlji tip. U stanicama hepatocelularnog karcinoma s utišanim AHCY smanjena je proliferacija i migracija te su aktivirani stanični putevi koji signaliziraju oštećenje DNA. Tijekom analize transkriptoma i proteoma fibroblasta pacijenta i stanica hepatocelularnog karcinoma s utišanim AHCY, uočena je moguća veza proteina AHCY s patologijom različitih neuroloških, jetrenih i mišićnih oboljenja, a predložena su i dva potencijalna biomarkera oboljenja.
Zaključci
Promjene razine AHCY mRNA i proteina, kao i lokalizacije AHCY u stanici koje su uočene u svih istraživanih mutanti proteina AHCY mogu biti odgovorne ili pridonositi patologiji kod oboljenja nedostatka AHCY. Prema predloženom mehanizmu AHCY ima utjecaj na oštećenje DNA i stanični ciklus. Dva potencijalna biomarkera su predložena s ciljem nastavka njihovog budućeg istraživanja u svrhu prognostike i dijagnostike oboljenja nedostatka AHCY.
Introduction and aims
S-adenosylhomocysteine hydrolase (AHCY) deficiency is a disorder caused by lowered enzymatic activity of AHCY protein due to the mutations in ahcy gene. AHCY has a key role in proper functioning of the methionine cycle in cell, therefore the lack of AHCY function causes severe metabolic disorder. Since AHCY is a single enzyme that hydrolyses SAH, a strong inhibitor of cellular metiltransferazes, it has a central role in maintaining the methylation status of the cell. Clinical presentation of this potentially lethal disorder includes a combination of muscular, neurological and hepatic disorders. Despite the essential activity of AHCY, changes in molecular and cellular mechanisms in the state of AHCY deficiency have so far been poorly investigated. The aim of this doctoral thesis is the analysis and understanding of molecular and cellular roles of AHCY and prediction of potential disease biomarkers.
Materials and methods/methodological approach
In this thesis, we performed studies of newly discovered Y328D mutant confirmed in the recent case of AHCY deficiency. Patient fibroblasts were obtained from the mentioned case, however, due to the limitations in experimental work with primary fibroblasts, we prepared, validated and investigated model cellular systems by combining methods ranging from classical biology approach to different omics and high throughput techniques.
Results
A method that enables visualization of protein interactions in human cells based on fluorescence of two complemented parts of Venus protein fused to proteins of interest was used for the first time to research AHCY protein. It is proven that Y328D mutant lacks the ability to form homodimes in human cells, and the method has been also successfully adapted to high throughput screening of AHCY protein interactors. Further, we report lower levels of mutant AHCY mRNA and protein in patient fibroblasts as well as cells that stably express AHCY mutant proteins. Microscopy revealed higher or lower amounts of mutant AHCY protein in cell nuclei in mentioned cells when compared to wild type. In hepatocellular carcinoma cells with a silenced AHCY expression we report lowered proliferation and migration as well as activation of DNA damage induced cellular pathways. While analyzing the transcriptome and proteome of patient fibroblasts and hepatocellular carcinoma cells with silenced AHCY expression, potential implications of AHCY protein in the pathology of various neurological, hepatic and muscular diseases are recognized, and two potential disease biomarkers are suggested.
Conclusions
Changes in levels of mutant AHCY mRNA and protein, as well as changed cellular localization of mutant AHCY could be responsible for or contribute to the pathology of the AHCY deficiency. As proposed by our mechanism, AHCY can imapact cellular DNA damage and cell cycle. Two biomarkers are suggested for further research and usage in prognostics and diagnostics of AHCY deficiency.
U miševa, prijenosnik natrija i glukoze (mSglt1/Slc5a1) apsorbira glukozu u četkastim membranama enterocita tankog crijeva i reapsorbira glukozu u četkastim membranama proksimalnih kanalića bubrega. ...Budući je stanična lokalizacija mSglt1 u drugim organima/tkivima neistražena, ekspresija mSglt1 određena je na razini mRNA i proteina usporedbom miševa divljeg tipa (Sglt1+/+) i Sglt1 knockout miševa (Sglt1-/-). Relativna ekspresija mRNA za mSglt1 određena qRT-PCRom najveća je u tankom crijevu; velika u sjemenskim vezikulama, bubrezima i žlijezdama slinovnicama (doušna, podčeljusna, podjezična); osrednja u prostati, jeziku, očima i maternici; i mala u gušterači, plućima i jetri. Imunofluorescencijskom analizom utvrđena je precizna stanična lokalizacija mSglt1 proteina u bubrezima, tankom crijevu, jetri, gušterači, žlijezdama slinovnicama, jeziku, prostati, bulbouretralnoj žlijezdi, sjemenskim vezikulama i maternici. U bubrezima miševa utvrđene su spolne razlike u ekspresiji mSglt1 na razini mRNA i proteina. Distribucija mSglt1 u različitim tkivima/organima miševa, kao učestalo korištenih pokusnih životinja, omogućava proučavanje njegove uloge u pato/fiziološkim, farmakološkim i toksikološkim istraživanjima.
In mice, sodium-glucose cotransporter (mSglt1/Slc5a1) absorbs and reabsorbs the glucose in the brush-border membrane of small intestinal enterocytes and renal proximal tubules, respectively. Due to unknown localization of mSglt1 in other organs/tissues, mSglt1 expression was determined comparing wild type (Sglt1+/+) and Sglt1 knockout mice (Sglt1-/-) at the mRNA and protein level. By qRT-PCR, mSglt1 mRNA expression was observed the highest in the small intestine; high in seminal vesicles, kidneys, salivary glands (parotid, submandibular, sublingual); medium in prostate, tongue, eyes and uterus; and small in pancreas, lung and liver. Immunofluorescence revealed detailed mSglt1 cell localization in the kidneys, small intestine, liver, pancreas, salivary glands, tongue, prostate, ulbourethral gland, seminal vesicles and uterus. In the kidneys, sex-dependent expression of mSglt1 was observed at mRNA and protein level. mSglt1 distribution in various mouse organs/tissues enables further studies of its role in patho/physiological, pharmacological and toxicological investigation in the most used experimental animals.
Cilj ovog istraživanja bio je usporediti ekspresiju citokina na razini gena i proteina u akutnoj i kroničnoj fazi infekcije HIV-om-1, te analizirati učinak dugotrajne supresije virusne replikacije u ...periodu duljem od dvije godine na citokinsku imunost. U dio istraživanja koji se bavi usporedbom citokinske imunosti na proteinskoj razini u akutnoj i kroničnoj fazi infekcije uključila sam 34 ispitanika, dok sam u istraživanje učinka dugotrajne supresije virusne replikacije na ekspresiju citokina u infekciji HIV-om-1 uključila 80 ispitanika. Ekspresiju 84 citokinska gena analizirala sam u tri ispitanika u akutnoj i tri u kroničnoj infekciji HIV-om-1 metodom „PCR array“. Za kvantifikaciju Th1/Th2/Th9/Th17/Th22 citokina primjenila sam citometriju pomoću kuglica (bead-based cytometry). U kroničnoj infekciji HIV-om-1 dokazala sam statistički značajno povećanje ekspresije 13 citokinskih gena (cd40lg, csf2, ifna5, il12b, il1b, il20, lta, osm, spp1, tgfa, tnfsf 11,14 i 8), te sniženje ekspresije il12a. U akutnoj fazi je u odnosu na kontrolnu skupinu došlo do povećanja koncentracija IL-10, IL-4 i TNF-α. Povećane koncentracije IL-10 i TNF-α, te uz njih IL-2, IL-6, IL-13 i IL-22 otkrivene su u kroničnoj infekciji u odnosu na kontrolu. Usporedbom ekspresije citokina između dviju faza infekcije HIV-om-1 vidljivo je smanjenje koncentracija IL-9. Koncentracija IL-17A snižena je u ispitanika prije primjene antiretrovirusne terapije, a kod kojih je dokazana supresija virusne replikacije. Ekspresija aktivacijskih biljega CD38 i HLA-DR snižena je u kroničnoj u odnosu na akutnu infekciju, te u supresiji virusne replikacije u odnosu na uzorke prije primjene terapije. U ovom je istraživanju dokazana promjena citokinskih profila u infekciji HIV-om-1 između akutne i kronične faze na razini genske i proteinske ekspresije.
The aim of this study was to compare cytokine expression on both gene and protein levels in acute and chronic phase of HIV type 1 infection and to analyze the effect of long-term suppression of viral replication in period longer than 2 years on cytokine immunity. To analyze cytokine expression on protein level in acute and chronic phase of HIV type 1 infection 34 patients were enrolled and the effect of long-term suppression of viral replication was determined in 80 HIV-positive patients. Using PCR array technology, expression of 84 cytokine genes was measured in 3 patients in acute and 3 patients in chronic phase of HIV type 1 infection. Bead-based cytometry was used to quantify levels of Th1/Th2/Th9/Th17/Th22 cytokines. The results showed statistically significant increase of 13 cytokine gene expression (cd40lg, csf2, ifna5, il12b, il1b, il20, lta, osm, spp1, tgfa, tnfsf 11, 14 and 8) and down regulation of the il12a expression in chronic HIV type 1 infection. Concentrations of IL-10, IL-4 and TNF-α were increased in the acute HIV type 1 infection when compared to control group. During chronic HIV type 1 infection there was an increase of IL-10, TNF-α, IL-2, IL-6, IL-13 and IL-22 levels when compared to control group. Comparison of cytokine expression between two stages of infection showed significant decrease in IL-9 concentration. Level of IL-17 was lower in therapy-naive patients with a suppression of viral replication. Expression of activation markers CD38 and HLA-DR was lower in a chronic stage of infection compared to acute infection as well as in samples with suppression of viral replication compared to samples before therapy initiation. This study showed change in cytokine profile in both gene and protein expression in different stages of HIV-infection.
Ova studija je osmišljena kako bi procijenili utjecaj oksidativnog stresa na extrapituitarnu sekreciju hormona rasta (GH) u oku, kao i analizirali međuovisnost razina produkcije očnog i serumskog GH ...u normalnim i uvjetima hipoksije. Ispitanici koji su predstavljali model oksidativnog stresa su 32 pacijenata s razvijenom proliferativnom dijabetičkom retinopatijom (PDR) i bili su liječeni kirurški pars plana vitrektomija (PPV). 49 pacijenata, ne-dijabetičara u kontrolnoj skupini (K) su izabrani između onih koji nisu patili od metaboličke bolesti, a ipak su morali biti operirani zbog drugih očnih bolesti. Uzorci vitreusa uzimani su tijekom PPV kirurškog zahvata, a istovremeno je uzeta i krv iz kubitalne vene. Mjerenja GH vrijednosti u serumu i vitreusu provedena su testom elektrokemijske luminiscencije (ECLIA). Procjena oksidativnog stresa izvedena je mjerenjem produkata uznapredovale oksidacije proteina (AOPP) i lipidne peroksidacije (LPO) pomoću testa enzimski vezane imunoabsorpcije (ELISA) u serumu i vitreusu. Serumske vrijednosti AOPP su bile značajno više nego vitrealne u obje skupine (p <0,001 za svaku skupinu), a za LPO samo u PDR skupini (p <0,001). Postoji značajna pozitivna korelacija između serumskih i vitrealnih vrijednosti LPO u skupini dijabetičara (r = 0,909, p <0,001).Vrijednosti GH u obije skupine značajno su veće u serumu, nego vitreusu (p <0,001). Usporedbom vrijednosti GH u serumu između kontrolne i PDR skupine, značajno više vrijednosti su pronađene kod dijabetičara (p = 0,012). Vitrealne vrijednosti GH izmjerene u obje skupine su slične, nešto više u dijabetičara. Rezultati našeg istraživanja su potvrdili postojanje sekrecije GH u oku i otkrili da je njegova produkcija u oku samostalna i neovisna o GH hipofize, a također je neovisna o utjecaju oksidativnog stresa.
This study was designed to evaluate influence of oxidative stress environment on extrapituitary growth hormone (GH) secretion in the eye as well as to analize interdependence between eye GH levels and serum GH levels in normal and hypoxic conditions. 32 patients with developed proliferative diabetic retinopathy (PDR) represented oxidative stress model and were treated surgically by pars plana vitrectomy (PPV). 49 patients, non-diabetics in control group were selected from those who have not suffered from metabolic diseases and yet they had to be operated due to other ocular disorders. Vitreous samples were taken during PPV surgical procedure and simultaneously blood from the cubital vein was collected. Measurements of GH values in serum and vitreous samples were conducted by electrochemical luminescence assay (ECLIA). Evaluation of oxidathive stress was carried out using enzyme-linked immunosorbent assay (ELISA) of Advanced Oxidation Protein Products (AOPP) and Lipide Hydroperoxide (LPO) in serum and vitreous as well. Serum values of AOPP were significantly higher than vitreal in both groups (p<0,001 for each group), and for LPO only in PDR group (p<0,001). Significant positive correlation between serum and vitreous LPO values in diabetic group (r=0,909; p<0,001) was observed. GH values in both groups were significantly higher in serum, than vitreous (p<0,001 for each group) and comparing GH serum values between control and PDR group, significantly higher amounts were found in diabetics (p=0,012). Vitreous GH values measured in both groups were similar, slightly higher in diabetics. Results of our study have confirmed existence of GH secresion in the eye and disclosed that GH production in eye is autonomous and independent to pituitary GH and is also independent to oxidative stress influence.
Osteoporoza je bolest kostura karakterizirana smanjenjem koštane mineralne gustoće (BMD) i poremećajem mikroarhitekture koštanog tkiva. Rizik za osteoporozu može biti genetski ili stečeni. Stečenom ...riziku pridonose brojni lijekovi, uključujući 13-cis retinoičnu kiselinu (13cRA). U ovome radu dokazano je da 13cRA u dozi od 80 mg kg-1 tijekom 14 dana uspješno uzrokuje osteoporotske promjene u štakora putem mehanizama povećanog oksidativnog stresa (OS) i stvaranja reaktivnih radikala, smanjenja aktivnosti osteoblasta i povećanja aktivnosti osteoklasta, smanjenja razine estrogena i pojavnosti upalnih citokina koji aktiviraju osteoklaste i uzrokuju apoptozu osteoblasta, smanjenja aktivnosti receptora vitamina D, smanjenja apsorpcije Ca2+ u crijevima i povećanja izlučivanja Ca bubrezima i učincima na paratireoidni hormon. Kroz model osteoporoze prouzročen 13cRA (RMO) u ovom istraživanju prikazana su protuoksidativnna, protuupalna, fitoestrogenska i regenerativna svojstva flavonoida (krizina, proantocijanida, naringenina, ikarina i kvercetina ) na osteoblaste i inhibiciju osteoklasta, što je pokazano odnosima između biljega OS, biokemijskim biljezima koštanog preokreta, mjerenjima koštane mase i geometrijskih odrednica bedrenih kosti, mjerenjima proupalnih citokina i histopatoloških oštećenja kosti. Učinci flavonoida uspoređeni su kroz navedene odrednice sa alendronatom, danas lijekom izbora u liječenju osteoporoze, a prikazana je i procjena oštećenja DNA kod RMO štakora nakon obrade flavonoidima i alendronatom. Dobiveni rezultati pokazuju mogućnost novog farmakoterapijskog pristupa u terapiji osteoporotičnih promjena u kojem laka dostupnost, ekonomska isplativost i nedostatak toksičnosti mogu biti prednost u primjeni flavonoida u odnosu na alendronat. Nadalje ovo istraživanje pridonosi boljem razumijevanju odnosa između prehrane i zdravlja te razvoja koncepta funkcionalne hrane s temeljnim ciljem postizanja boljeg zdravstvenog statusa, spriječavanja i liječenja bolesti.
Osteoporosis is a skeletal disease characterized by decreased bone mineral density (BMD) and bone microarchitecture disorder. The risk of osteoporosis can be genetic or acquired. Numerous medications including 13-cis retinoic acid (13cRA) contribute to the acquired risk. In this PhD thesis it has been proved that 13cRA at a dose of 80 mg kg-1 during 14 days successfully causes osteoporotic changes in the rats through the mechanisms of increased oxidative stress (OS) and the formation of reactive radicals, reduced activity of osteoblasts and increased osteoclast activity, decreased estrogen levels and the appearance of inflammatory cytokines which activate osteoclasts and cause osteoblasts apoptosis, reduction in vitamin D receptor activity, decreased absorption of Ca2+ in the intestine, increase the Ca secretion by kidneys and the effects on the parathyroid hormone. Through the model of osteoporosis caused by 13cRA (RMO) in this study are shown antioxidative, anti-inflammatory, phytoestrogenic and regenerative features of flavonoids (chrysin, proanthocyanidins, naringenin, icariin and quercetin) on osteoblasts and osteoclasts inhibition, which has been shown by the relationships between markers of OS, biochemical markers of bone turnover, measurements of bone mass and geometric determinants of thigh bones, measurements of proinflammatory cytokines and histopathological bone damage. The effects of flavonoids are compared through these features with alendronate, nowadays the medication of choice to treat osteoporosis, and the evaluation of DNA damage at RMO rats after the treatment with flavonoids and alendronate is presented. The results show the possibility of a new pharmacological approach for the treatment of osteoporotic changes, where easy accessibility, cost-effectiveness and lack of toxicity may be an advantage in the application of flavonoids with respect to alendronate. Furthermore, this study contributes to a better understanding of the relationship between diet and health and development of the concept of functional foods with the fundamental aim of achieving better health status, prevention and treatment of disease.
Većina psihoaktivnih vrsta gljiva sadrži psilocibin, psilocin, beocistin ili nor-beocistin, a zbog djelovanja psilocina sličnog LSD-u koriste se kao rekreacijska droga i zabranjene su. Dokazivanje ...aktivnih tvari iz forenzičkih uzoraka toksikološkim metodama GC-MS i LC-MS nije moguće iz otrusine spora ili micelija prije procesa fruktificiranja. Glavni cilj ovog rada bio je ukazati na potencijal primjene molekularnih i morfoloških metoda u forenzičkoj analizi psihoaktivnih gljiva. Morfološkom analizom 63 forenzička uzorka uspješno je identificirano 5 vrsta, a najzastupljenija je vrsta Psilocybe cubensis. Sekvenciranjem cijele regije ITS i dijela gena LSU forenzičkih uzoraka potvrđeni su rezultati morfološke analize i uspješno je identificirana šesta vrsta iz otrusine spora. Obje metode mogu se implementirati u rutinsku forenzičku analizu uzoraka gljiva. Potencijal istraživanog modela preliminarne identifikacije psihoaktivnih gljiva korištenjem specifičnih sondi TaqMan®MGB u reakciji Real Time PCR nije potvrđen. Analizom LC-MS otkriven je psihoaktivni status vrsta Stropharia semiglobata i Galerina badipes.
Most of psychoactive fungi contain psilocybin, psilocin, baeocystin or nor- baeocystin, and due to effects of psilocin, which are similar to LSD, they are illegal. Proving active substances from forensic samples through toxicological methods is not possible from spore print or from mycelia. Main goal of this thesis was to emphasize the potential of using molecular and morphological methods in forensic analysis of psychoactive fungi. Morphological analysis of 63 forensic samples resulted with 5 successfully identified species with Psilocybe cubensis as the most common. Through sequencing the ITS region and the part of LSU gene of forensic samples, morphological analysis results were confirmed and sixth species was successfully identified from spore print. Both methods can be implemented in the routine forensic workflow for fungi. Potential of the researched model for preliminary identification of psychoactive fungi by using specific TaqMan®MGB probes in RT-PCR reaction, was not confirmed. LC-MS analysis established the psychoactive status of species Stropharia semiglobata and Galerina badipes.
U ovom su radu ispitane fitokemijske značajke, antimikrobna svojstva i biološki učinci lista obične planike. Određen je sadržaj ukupnih fenola, flavonoida, tanina, fenolnih kiselina, te polifenolni ...profil lista UHPLC-MS/MS (Orbitrap) metodom, sadržaj arbutina i hidrokinona HPLC-UV/DAD metodom, antioksidacijska svojstva FRAP, TEAC i DPPH metodama. MIK koncentracije su utvrđene dvostrukom mikrodilucijom, a mutagenost komet-testom i CBMN testom. Dokazano je 37 novih fenolnih spojeva. Sadržaj arbutina u listu bio je 0,3 %, dok hidrokinon nije detektiran. Metanol je bolje sredstvo za ekstrakciju polifenola od vode. Metanolni ekstrakt lista pokazao je bolji antioksidacijski, te antimikrobni učinak na ATCC i kliničke sojeve mikroorganizama. Arbutin nije pokazao antimikrobni učinak, dok je njegov metabolit hidrokinon pokazao pozitivan učinak na ispitivane uropatogene. U uvjetima in vitro i in vivo vodeni ekstrakt lista pri koncentraciji arbutina koja odgovara maksimalno dopuštenom dnevnom unosu ima nisku genotoksičnost. Potencijal vodenog ekstrakta lista obične planike kao fitoterpeutika za kliničku primjenu je velik.
This thesis investigated the phytochemical characteristics, antimicrobial properties and biological effects of strawberry tree leaves. Total phenol, tannin, flavonoid and phenolic acid content was determined as well as polyphenolic profile using the UHPLC-MS/MS Orbitrap method. Arbutin and hydroquinone concentrations were measured by HPLC-UV/DAD, antioxidative properties by FRAP, TEAC, DPPH, MIC concentrations by a Twofolded Microdilution method and mutagenicity by Comet-test and CBMN test. Thirty-seven new phenolic compounds were detected. Leaves contained 0.3% of arbutin while hydroquinone was not detected. Methanol was shown to be a more effective solvent for the extraction of polyphenols. Methanolic extract showed better antioxidative properties and better antimicrobial effect on standard ATCC and clinical microorganism strains. Arbutin did not show antimicrobial activity, while its metabolite hydroquinone showed positive antimicrobial activity against the tested uropathogens. Water extract applied at an arbutin concentration corresponding to the maximum allowable daily intake showed low genotoxicity in vitro and in vivo. Strawberry tree water leaf extract has great potential as a phytotherapeutic in clinical application.
Koštani morfogenetski protein 6 (BMP6) je ključni regulator hepcidina, proteina koji sudjeluje u održavanju homeostaze željeza. Istražili smo utjecaj željeza na BMP6 u tkivima i plazmi, te zašto ...ostale BMP molekule nisu u mogućnosti spriječiti hematokromatozu u Bmp6 -/- miševa. Kod miševa divljeg tipa, 4 sata nakon tretmana željezom dolazi do povećanja izražaja gena za BMP6 i hepcidin u jetri, dok je izražaj ostalih BMP molekula nepromijenjen. BMP6 je detektiran u cirkulaciji miševa i štakora, te je pružen prvi dokaz utjecaja željeza na povećanje razine BMP6 u plazmi. Naprotiv, u Bmp6 -/- miševa, tretman željezom dovodi do zakašnjele i slabije aktivacije Smad signalnog puta i sinteze hepcidina u jetri nakon 24 sata, zajedno s povećanim izražajem gena za BMP2, 4, 5 i 9 u duodenumu i BMP5 i BMP7 u jetri. Povećana sinteza BMP7 u jetri potencijalno doprinosi indukciji izražaja gena za hepcidin, što je dodatno podržano BMP7 terapijom koja dovodi do djelotvorne sinteze hepcidina i normalizacije serumskog željeza. No, sinteza BMP7 nije povezana s metabolizmom željeza, već s uzročnim oštećenjima jetre gdje BMP7 predstavlja mogući oblik obrambenog mehanizma od viška nakupljenog željeza. Rezultati pokazuju da akutna doza željeza aktivira kompenzacijske mehanizme s različitim BMP molekulama, ali nedovoljnog intenziteta da bi se spriječila hematokromatoza u Bmp6 -/- miševa. Učinkovita terapija s BMP7 sugerira da je BMP6 bitan, ali ne isključiv regulator homeostaze željeza in vivo.
Bone morphogenetic protein 6 (BMP6) is a key regulator of hepcidin expression, a key iron regulatory hormone. We investigated the effect of iron on Bmp6 expression in tissues and plasma, and why other BMPs could not prevent hemochromatosis in Bmp6 -/- mice. In WT mice, 4 hours following iron challenge liver Bmp6 and Hamp expression were increased, while expression of other Bmps was not affected. In parallel, we provided the first evidence that iron increased the circulating BMP6 level. On the contrary, in Bmp6 -/- mice, iron challenge lead to a 24 hours delayed and blunted activation of liver Smad signaling and hepcidin expression, that was accompanied by an increased expression of Bmp2, 4, 5 and 9 in the duodenum and Bmp5 and Bmp7 in the liver. High liver Bmp7 expression potentially contributed to the late hepcidin response, which was further supported by BMP7 therapy resulting in an effective hepcidin expression and normalization of serum iron. However, BMP7 synthesis was not associated with iron metabolism, but the causal hepatic injuries where BMP7 represented a form of defense mechanism to the excessive iron accumulation. Our data indicate that iron activated BMP compensatory mechanisms that were not sufficient for preventing hemochromatosis in Bmp6 -/- mice. However, the effective BMP7 therapy suggested that BMP6 is an essential but not exclusive in vivo regulator of iron homeostasis.
