Kronična opstrukcijska plućna bolest (KOPB) je složeni poremećaj koji zahvaća pluća, ali
i sistemski odjeljak. Naše se istraživanje temeljilo na hipotezi da je u bolesnika s KOPB-om
prisutna ...sistemska upala i sistemski oksidacijski stres. Sukladno tome, cilj ovog istraživanja
bio je istražiti sistemske pokazatelje oksidacijskih i upalnih promjena u bolesnika sa stabilnim
KOPB-om te ispitati povezanost sistemskih antioksidansa s poremećajem funkcije pluća i
biljezima sistemske upale. Ispitali smo i dijagnostičku učinkovitost biljega sistemskog
oksidacijskog stresa u razlikovanju zdravih i oboljelih od KOPB-a.
Sistemski biljezi oksidacijskog stresa (albumin, transferin, ceruloplazmin, ukupni bilirubin,
slobodne tiolne skupine, malondialdehid (MDA), paraoksonazna i arilesterazna aktivnost
paraoksonaze 1 (PON1), te ekspresija i aktivacija unutarstaničnih signalnih molekula Hsp27,
Hsp70, ERK, JNK i p38) određeni su u 106 bolesnika sa stabilnim KOPB-om i 45 zdravih
ispitanika. Ispitane su i njihove povezanosti s biljezima sistemske upale (CRP, fibrinogen,
ukupni leukociti), pušenjem i pokazateljima funkcije pluća (FEV1 i FEV1/FVC).
Bolesnici s KOPB-om su imali povišene koncentracije ceruloplazmina i MDA, snižene
koncentracije albumina, transferina i tiola, te snižene obje ispitane aktivnosti PON1.
Koncentracije ukupnog bilirubina nisu se razlikovale usporedbom ispitivanih skupina.
Ceruloplazmin je pokazao pozitivnu korelaciju s CRP-om i fibrinogenom. Albumin i
transferin su pokazali negativnu korelaciju s CRP-om, te pozitivnu korelaciju sa slobodnim
tiolima. Transferin je negativno korelirao s fibrinogenom. Jedini pokazatelj povezan s
funkcijom pluća bio je MDA. Usporedbom pušača, bivših pušača i nepušača iz skupine
bolesnika s KOPB-om nisu nađene razlike u koncentracijama promatranih biljega
oksidacijskog stresa i upale. Povišene koncentracije ceruloplazmina su najsnažniji prediktor
prisutnosti KOPB-a. Model koji uključuje ceruloplazmin, albumin, MDA i arilesteraznu
aktivnost PON1 te biljege sistemske upale pokazao je najbolju dijagnostičku učinkovitost u
predviđanju KOPB-a (AUC (95%CI) = 0,96 (0,92 – 0,99)). Predloženi bi model mogao
ispravno predvidjeti prisutnost KOPB-a u 89% bolesnika. Također, naši su rezultati ukazali da
je razina ekspresije Hsp27 i Hsp70 u leukocitima periferne bila je najniža u pušača s KOPBom.
Ekspresija svih ispitivanih MAPK (ERK, JNK i p38) u perifernim leukocitima bolesnika
s KOPB-om nije se razlikovala u usporedbi sa zdravim ispitanicima. Aktivacija ERK bila je
značajnija kod zdravih i bolesnih nepušača, dok je aktivacija JNK i p38 bila najizraženija u
pušača s KOPB-om.
Rezultati su ukazali da prisutnost bolesti i pušenje utječu na ispitane unutarstanične
signalne molekule. Bolje razumijevanje ovih molekularnih mehanizama moglo bi pomoći u
dijagnozi i pronalaženju novih terapijskih meta za KOPB. Dijagnostičke karakteristike
predloženog modela koji kombinira koncentracije biljega sistemske upale i sistemskog
oksidacijskog stresa ukazuju da bi se on mogao koristiti kao vrijedan alat u razlikovanju
zdravih ispitanika i bolesnika s KOPB-om.
Background: Chronic obstructive pulmonary disease (COPD) is a complex disorder
affecting the lungs and the systemic compartment. We hypothesized that systemic
inflammation and systemic oxidative stress are present in patients with COPD. Therefore, we
aimed to investigate markers of systemic oxidative and inflammatory alterations in patients
with stable COPD, and to test the association of systemic antioxidants with indicators of lung
function and systemic inflammation. The diagnostic accuracy of systemic oxidative stress
parameters in distinguishing between healthy subjects and patients with COPD was also
evaluated.
Materials and methods: Systemic oxidative stress markers (albumin, transferrin,
ceruloplasmin, total bilirubin, thiols, malondialdehyde (MDA), paraoxonase and arylesterase
activity of paraoxonase 1 (PON1), and the expression and activation of intracellular signalling
molecules Hsp27, Hsp70, ERK, JNK i p38) were assessed in 106 stable COPD patients and
45 healthy subjects. Their association with systemic inflammatory markers (CRP, fibrinogen,
total leukocytes), smoking status and lung function parameters (FEV1 i FEV1/FVC) was
investigated.
Results: Higher ceruloplasmin and MDA concentrations, and lower albumin, transferrin,
thiols and PON1 activities (paraoxonase and arylesterase) were found in patients with COPD.
Total bilirubin concentrations were similar in the studied groups. Ceruloplasmin showed a
positive correlation with CRP and fibrinogen. Albumin and transferrin showed a negative
correlation with CRP, and a positive corelation with thiols. Transferrin negatively correlated
with fibrinogen. Only MDA showed an association with pulmonary function. No differences
were found comparing concentrations of oxidative stress and inflammatory markers between
COPD patients subdivided according to their smoking status. Ceruloplasmin was the strongest
single predictor of COPD. The model combining ceruloplasmin, albumin, MDA, arylesterase
PON1 activity, and markers of systemic inflammation demonstrated very good diagnostic
performances (AUC (95%CI) = 0,96 (0,92 – 0,99)). The proposed model correctly identifies
89% of patients with COPD. In addition, our results showed that the decrease in expression of
peripheral blood leukocytes' Hsp27 and Hsp70 was the most prominent in COPD smokers.
Expression levels of all three MAPKs investigated was not altered in leukocytes of COPD
patients compared to healthy subjects. However, ERK activation was stimulated in healthy and COPD non-smokers, while JNK and p38 activation was the most pronounced in COPD
smokers.
Conclusions: Our results showed that COPD and smoking affect the intracellular
signalling pathways investigated. Improved understanding of these molecular mechanisms
could help identify novel targets for diagnosis and therapeutic interventions in COPD.
Diagnostic characteristics of the proposed model, obtained by combining markers of systemic
inflammation and systemic oxidative stress, suggest its potential value as an additional tool in
COPD diagnosis.
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.
Kardiovaskularne bolesti uzrokovane aterosklerozom predstavljaju značajan uzrok smrtnosti u hemodijaliziranih
(HD) bolesnika. Bolesnici koji se liječe HD imaju i do 30 puta veću stopu smrtnosti ...uzrokovanu kardiovaskularnim
bolestima. Rizični čimbenici uključeni u patogenezu ateroskleroze u HD bolesnika pacijenata uključuju dijabetes,
hipertenziju, dislipidemiju, pušenje, oksidativni stres, upalu te endotelnu disfunkciju. Budući da ateroskleroza ima
dugačku subkliničku fazu veoma je važno da patološki procesi budu otkriveni čim ranije, dok je bolest još u
asimptomatskoj fazi. Povećana debljina intime-medije (IMT) prepoznata je kao rani indikator subkliničke
ateroskleroze, kako u općoj populaciji tako i u HD-ih bolesnika te je povezana s tradicionalnim i novijim
kardiovaskularnim čimbenicima rizika. Nekoliko je studija dokazalo da je karotidna ateroskleroza povezana s
adhezijskim molekulama te omentin-1 proteinom. Ciljevi ovog rada bili su sljedeći: a) istražiti povezanost sICAM-1,
sVCAM-1, omentin-1 proteina te ostalih netradicionalnih rizičnih čimbenika sa subkliničkom aterosklerozom; b)
ispitati dijagnostičku vrijednost ovih specifičnih markera u otkrivanju subkliničke ateroskleroze i c) ispitati njihovu
ulogu kao prediktora smrtnosti u asimptomatskih bolesnika.
Počevši od studenog 2011. godine, kohorta od 210 HD-ih bolesnika sudjelovala je u trogodišnjem istraživanju.
Ispitanici su bili podijeljeni u tri skupine ovisno o prisutnosti ateroskleroze. Ateroskleroza je dijagnosticirana na
temelju mjerenja debljine intima-medija karotidne arterije. Uzorkovanje krvi izvršeno je na početku studije te svakih
12 mjeseci iza toga sve do kraja istraživanja. Ehokardiografska mjerenja učinjena su na početku i na kraju studije.
Dokazana je slaba korelacije između IMT i sICAM-1 (r=0,39, P=0,001), sVCAM-1 (r=0,27, P=0,015) i omentin-1
proteina (r=-0,25, P=0,020). Omentin-1 protein je pokazao dobru korelaciju s parametrima sistoličke i dijastoličke
funkcije lijeve klijetke (r=0,52, P=0,001 i r=0,51, P=0,001). Multivarijantna analiza je pokazala kako su koncentracije
sICAM-1 i omentin-1 proteina snažni neovisni pretkazatelji IMT (P=0,031 i P=0,010). Coxova analiza
proporcionalnih rizika pokazala je da su koncentracija sICAM-1 i omentin-1 proteina snažni pretkazatelji
kardiovaskularne smrti (HR=1,85, CI=1,18-2,32, P=0,021 i HR=4,14, CI=1,38-12,1, P=0,004,) te da uzastopno
mjerenje koncentracija sICAM-1 i omentin-1 proteina predstavlja jake prediktore IMT progresije (HR=1,98, 95%
CI=1,21-2,38, P<0,002 i HR=2,91, 95% CI=1,57-4,72, P<0,001).
Naša studija je pokazala kako su koncentracije ICAM-1 I omentin-1 proteina jaki pretkazatelji kardiovaskularne
smrti u asimptomatskih HD bolesnika. Porast koncentracije ICAM-1 te pad koncentracije omentin-1 proteina imaju
ključnu ulogu u ranoj progresiji ateroskleroze.
Atherosclerotic cardiovascular complications represent significant cause of mortality in hemodialysis (HD)
patients. Patients undergoing HD have up to 30 times higher incidence of cardiovascular disease mortality. Risk
factors involved in pathogenesis of atherosclerosis in patients undergoing HD include diabetes, hypertension,
dyslipidemia, smoking, oxidative stress, inflammation and endothelial dysfunction. Since atherosclerosis has a long
subclinical phase it is important that pathogenic processes are identified while disease is still asymptomatic. Increased
carotid intima-media thickness (cIMT) is well recognized as an early indicator of subclinical atherosclerosis both in
the general population and in HD patients and it is associated with traditional and emerging CV risk factors. Several
studies have shown that carotid atherosclerosis is associated with circulating concentrations of soluble adhesion
molecules (CAMs) and omentin-1 protein. The aims of this study were to: a) investigate association of sICAM-1,
sVCAM-1, omentin-1 and other non-traditional risk factors with subclinical atherosclerosis; b) examine the diagnostic
value of these specific markers in the early detection of subclinical atherosclerosis and c) examine their role as
predictors of mortality in group of patients with subclinical atherosclerosis on regular HD.
Starting from November 2011, a cohort of 210 HD patients participated in this three-year follow-up study. The
subjects were divided into three groups according to the presence of atherosclerosis. Atherosclerotic disease was
assessed by measuring carotid intima-media thickness (IMT). Samplings were withdrawn at baseline and thereafter
every 12 months until the end of follow-up. Echocardiography was performed at baseline and at the end of follow-up.
IMT showed weak correlation with sICAM-1 (r=0.39, P=0.001), sVCAM-1 (r=0.27, P=0.015), and omentin-1 (r=-
0.25, P=0.020), also omentin-1 showed good correlation with parameters of systolic and diastolic function (r=0.52,
P=0.001 and r=0.51, P=0.001). Multivariate analysis showed that sICAM-1 and sVCAM-1 concentrations were a
strong independent correlate of IMT (P=0.031 and P=0.010, respectively). The Cox proportional analysis showed that
sICAM-1 and omentin-1 concentrations were strong predictors of cardiovascular death (HR=1.85, CI=1.18-2.32,
P=0.021 and HR=4.14, CI=1.38-12.1, P=0.004, respectively) and that serial measurements of these markers predict
IMT progression (HR=1.98, 95% CI=1.21-2.38, P<0.002 and HR=2.91, 95% CI=1.57-4.72, P<0.001, respectively).
Our study demonstrated that sICAM-1 and omentin-1 levels are strong predictors of cardiovascular death in HD
patients with subclinical atherosclerosis. . We believe that an increase in sICAM-1 and decrease in omentin-1 levels
could play an important role in the early progression of atherosclerosis.
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.
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.
Moždani udar (MU) u djece heterogeni je poremećaj s višestrukom etiologijom, koja je još uvijek nepoznata u
približno 30% slučajeva. Genetički čimbenici nisu do kraja karakterizirani, a raspodjela ...gena kandidata za
nastanak MU različita je u pojedinim populacijama. U istraživanje je bilo uključeno 100 djece starosne dobi do
18 godina s potvrđenim MU i 100 zdrave djece podudarne po dobi i spolu djeci s MU. Napravljena je
genotipizacija 14 polimorfizama u 12 gena kandidata koji kodiraju proteine uključene u sustav zgrušavanja i
fibrinolize (FV Leiden, FV HR2, FII G20210A, beta-fibrinogen -455G>A, FXIII-A Val34Leu, PAI-1 4G/5G),
ljudskih trombocitnih antigena (HPA-1, -2, -3 i -5), metabolizma homocisteina (MTHFR C677T, MTHFR
A1298C) i intermedijernih rizicnih cimbenika (ACE I/D i ApoE 2-4). Utvrđena je cešća pojavnost MU u
dječaka, koja je uočena i u svim ispitivanim podskupinama. Najveći rizik za nastanak MU zabilježen je u prvoj
godini života, kao i 2,7 puta veći broj arterijskih ishemijskih MU (AIMU) u odnosu na hemoragijski MU
(HMU). Rezultati genotipizacije potvrdili su prije opisanu povezanost FV Leiden s nastankom (pojavom) MU.
Dokazana je i povezanost 4 polimorfizma (FV Leiden, FXIII-A Val34Leu, HPA-3 i apoE _2-4) s AIMU i to
različitih polimorfizama u pojedinim podskupinama prema spolu i dobi pojave AIMU, kao i povezanost
kombinacija genotipova polimorfizama MTHFR C677T i MTHFR A1298 (CC/AC) i dvostrukog heterozigotnog
oblika (GA/AG) polimorfizama FV Leiden i FV HR2 s AIMU, te kombinacije genotipova GA/AA s
perinatalnim AIMU. Utvrđen je i umjereno povećan rizik za pojavu HMU u nosioca haplotipa HA2 (HPA-
1a2a3a-ACED). Ovo istraživanje ukazalo je na povezanost pojedinih dosad nedovoljno istraženih polimorfizama
kao i kombinacija polimorfizama s etiologijom MU u djece u Hrvatskoj, što upotpunjuje i proširuje dosadašnje
spoznaje o etiološkim rizičnim čimbenicima.
Although stroke in children is a relatively rare and heterogeneous disorder with a wide range of identified risk
factors, the etiology of stroke is still undetermined in up to 30% of children. Taking into consideration that
genetic risk factors are incompletely characterized at present and the frequency of genetic factors may vary
among different populations, a genotype analysis was perforrmed of 14 polymorphisms in 12 candidate genes
encoding proteins of the coagulation and fibrinolysis systems (FV Leiden, FV HR2, FII G20210A, beta-fibrinogen -
455G>A, FXIII-A Val34Leu, PAI-1 4G/5G), human platelet alloantigens (HPA-1, -2, -3 and -5), homocysteine
metabolism (MTHFR C677T, MTHFR A1298C) and intermediate risk factors (ACE I/D and apoE 2-4) was
performed. The subject group comprised 100 children with a confirmed diagnosis of stroke aged £18 years and
100 age- and sex-matched control subjects. Obtained results were also analyzed in gender-specific stroke group
and subgroups according to the type of stroke: arterial ischemic (AIS) and hemorrhagic stroke (HS), and
according to the time of stroke onset (perinatal and childhood AIS). The predominance of boys was found
among (in) children with stroke and in all tested subgroups. The greatest risk for stroke was identified in the first
year of life, and AIS was found 2.7 times more frequently than HS. This case-control study has confirmed the
association between FV Leiden and stroke that was also observed in numerous studies so far, but it has also
shown that other previously not reported polymorhisms (FXIII-A Val34Leu, HPA-3 and the combination of FV
Leiden and FV HR2 polymorphisms) can be related to AIS in Croatian population. Analysis performed in
gender-specific stroke subgroups revealed the association of different polymorphisms in boys (FXII-A
Val34Leu, HPA-3 and combination of MTHFR polymorphisms) and girls (apoE 4 allele). The strongest
association (with OR>10.0) was found between FV Leiden and perinatal AIS for both genders whereas the
lowest risk for perinatal AIS was observed in girls who were carriers of at least one HPA-3b allele. Obtained
results have at least partially elucidated the role (impact) of new evaluated polymorphisms in the etiology of
stroke in children and their impact according to gender, type of stroke and time of stroke onset.
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.
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.
