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Synthesis and characterization of amides of 3- and 6- substituted β-carboline derivatives and chloroquine analogs
No full textU ovom radu sintetizirana su i strukturno okarakterizirana dva nova hibridna spoja 4a i 4b, sastavljena od β-karbolinskog prstena koji je amidnom poveznicom preko položaja 3 i 6 povezan s kinolinskim derivatom po uzoru na klorokin.
Za sintezu ovih spojeva bilo je potrebno sintetizirati obje građevne jedinice, kinolinski derivat, (7-klorokinolin-4-il)glicin, i 3- i 6- supstituirane β-karbolinske amine 1e i 2e. Priprava amina 1e i 2e na položajima β-karbolinskog prstena polazi iz metilnog estera triptofana i 5-metoksitriptamina te se odvija u pet reakcijskih koraka. Najprije Pictet-Spenglerovom kondenzacijom dolazi do nastanka tetrahidro-β-karbolina (1a i 2a), koji se zatim oksidiraju do odgovarajućih β-karbolina 1b i 2b. U slučaju amina iz serije 1, metilni ester na položaju 3 se reducira do primarnog alkohola 1c. Slijedi konverzija alkoholne u azidnu skupinu uz pomoć donora azida ADMP-a u bazičnim uvjetima čime nastaje odgovarajući azid 1d, te njegova redukcija u primarni amin 1e.
Eter na položaju 6 spoja 2b hidrolizom u kiselom prelazi u odgovarajući fenol 2c, koji u reakciji nukleofilne supstitucije u bazičnim uvjetima prelazi u spoj 2d kod kojeg terminalna amino skupina posjeduje Boc-zaštitnu skupinu. Potom se amino skupina oslobađa zaštite, hidrolizom funkcionalne skupine karbamata u kiselom, te nastaje amin 2e.
Konačni produkti 4a i 4b dobiveni su u reakciji amina 1e i 2e s (7-klorokinolin-4-il)glicinom (3), koristeći standardne coupling uvjete (T3P, TEA, DMF).
Strukture sintetiziranih spojeva potvrđene su uobičajenim spektroskopskim/spektrometrijskim metodama (1H i 13C NMR, MS, IR). Sva četiri spoja zadovoljavaju Lipinskijeva i Veberova pravila, a dobru oralnu bioraspoloživost predviđa im i alat SwissADME. Sintetiziranim derivatima će u daljnjim istraţivanjima biti ispitano biološko djelovanje.In this work, two novel hybrid compounds 4a and 4b, consisting of a β-carboline ring which is connected to a quinoline derivative via the chloroquine model by an amide bond in positions 3 and 6, were synthesized and characterized.
In order to achieve this goal, building blocks for these compounds were prepared, a quinoline derivative (7-chloroquinolin-4-yl) glycine and 3- and 6-substituted β-carboline amines 1e and 2e. The preparation of amines 1e and 2e at the β-carboline ring positions starts from the methyl ester of tryptophan and 5-methoxytryptamine and takes place in five reaction steps. First, Pictet-Spengler condensation produces tetrahydro-β-carbolines (1a and 2a), which are then oxidized to the corresponding β-carbolines 1b and 2b. In case of amine from series 1, the methyl ester at position 3 is reduced to the primary alcohol 1c. This is followed by the conversion of the alcohol 1c to the corresponding azide 1d by using ADMP under basic conditions, and reduction to the primary amine 1e.
The ether moiety at the position 6 of the compound 2b is hydrolized in acidic conditions to corresponding phenol 2c, which in the reaction of nucleophilic substitution under basic conditions is converted to compound 2d in which the terminal amino group carries a Boc-protecting group. The amino group is then deprotected by acid hydrolysis of the carbamate, and amine 2e is formed.
The final products 4a and 4b were obtained by the reaction of amines 1e and 2e with (7-chloroquinolin-4-yl) glycine (3), by using standard coupling conditions (T3P, TEA, DMF).
Structures of the newly prepared compounds were confirmed using conventional analytical and spectroscopic methods (1H i 13C NMR, MS, IR). All four novel derivatives meet all of the Lipinski's and Veber's criteria for good oral bioavailability of potential drugs. SwissADME tool predicts good oral bioavailability of all four derivatives. Further studies will investigate biological activity of the prepared coumpounds
Vanillactic and vanilmandelic acid - new biomarkers in diagnosics of aromatic L-amino acid decarboxylase deficiency
No full textManjak aktivnosti dekarboksilaze aromatskih L-aminokiselina rijedak je autosomno-recesivni poremećaj koji
dovodi do teškog deficita kateholamina i serotonina. Klinička slika uključuje različite stupnjeve neuroloških oštećenja,
uključujući motoričke i neuromotoričke simptome. Uspješnost liječenja ovisi o pravovremenom postavljanju dijagnoze
i početku terapije. Postavljanje dijagnoze manjka aktivnosti AADC temelji se na određivanju koncentracije
neurotransmitera u likvoru i molekularnoj dijagnostici zbog čega se bolesnici izlažu invazivnom postupku uzimanja
uzoraka za analizu. Vanillaktat (VLA), vanilmandelična kiselina (VMA) i njihov omjer VLA/VMA mogu se
analizirati u sklopu rutinske analize organskih kiselina u urinu i smatraju se potencijalnim neinvazivnim biokemijskim
biljezima u postavljanju dijagnoze AADC. Cilj ovog diplomskog rada bio je odrediti referentne intervale za VLA,
VMA i njihova omjera VLA/VMA u urinu retrospektivnom analizom organskih kiselina bolesnika suspektnih na
neurometaboličke poremećaje. 219 obrađenih bolesnika podijeljeno je u tri dobne skupine, a uzorci su analizirani inhouse
metodom ekstrakcije organskih kiselina na sustavu za plinsku kromatografiju s masenom spektrometrijom. Kao
dodatni marker korišten je N-acetiltirozin. Određivanje referentnih intervala VLA, VMA i VLA/VMA u urinu u
sklopu rutinske analize organskih kiselina može pomoći u diferencijalnoj dijagnostici neurometaboličkih bolesti.Aromatic L-amino acid decarboxylase deficiency is rare autosomal recessive disorder that leads to severe deficit
of catecholamine and serotonin. The clinical picture includes varying degree of neurological damage, including motor
and neuromotor symptoms. Treatment outcomes depends on the timepoint when therapy started. Key diagnostic test
for AADC confirmation are measurement neurotransmiters in cerebrospinal fluid and genetic testing. However, to
perform this testing, patients are exposed to intensive lumbar puncture procedure. Determination of vanillactic acid
(VLA), vanillylmandelic acid (VMA) and VLA/VMA ratio as part of organic acid analysis in urine is considered a
reliable approach using non-invasive biomarkers in the diagnosis of AADC. The aim of this thesis was to determine
reference intervals of VLA, VMA and VLA/VMA ratio in urine. Retrospective analysis of urinary organic acids
obtained from 219 patients suspected for neurometabolic disorders were included in this study. Patients were divided
into groups depending on age. Organic acids in urine were measured using in-house method by gas-chromatography
mass spectrometry. As additional biomarker, N-acetyltyrosine was used. Determination of reference intervals of VLA,
VMA and VLA/VMA ratio as a part of organic acids analysis in urine may help to differentiate neurometabolic
patients with possible AADC deficiency from other neurometabolic disorders
Serological testing of hepatitis E virus infection at the Croatian Institute of Transfusion Medicine
No full textZa virus hepatitisa E (HEV) karakteristično je da ga većina ljudi preboli asimptomatski. Ipak, za određene skupine kao
što su trudnice, pacijenti na imunosupresivnoj terapiji te za imunokompromitirane pojedince s već postojećim bolestima
jetre, HEV infekcija može dovesti do letalnog ishoda.Tijekom 16 mjeseci (2019. i 2020. godine), nešto više od 500
pacijenata serološki je ispitivano na biljege hepatitisa E u Hrvatskom zavodu za transfuzijsku medicinu (HZTM), i to u
Odjelu za dijagnostiku krvlju prenosivih bolesti (OKB). Korišteni su komercijalni HEV IgG Dia.Pro i HEV IgM Dia.Pro
testovi koji se temelje na ELISA metodi. Ispitivanja su obavljena na Gemini analizatoru i kompletan protokol pratio je
upute proizvođača. Obzirom na rezultate testiranja pacijenata (TP), HEV IgG seroprevalencija iznosi 23,2 % (117
uzoraka) dok je stopa HEV IgM seroprevalencije 9,3 % (47 uzoraka). HZTM još je 2014. godine proveo
seroprevalencijsku studiju koja je obuhvatila 1036 uzoraka dobrovoljnih darivatelja krvi (DDK). HEV seroprevalencija u
DDK-a procijenjena je s četiri dostupna testa (3 ELISA i 1 imunoblot (IB) test). Uspoređeni su rezultati dobiveni Dia.Pro
testom. HEV IgM seroprevalencija među TP statistički je značajno veća u odnosu na DDK (9,3 % naspram 4,4 %) što je
dokazano hi-kvadrat testom (χ2 test) uz razinu značajnosti testa α=0,05 (χ2=15,13 > χ2
df=1,α=0,05=3,84, p < 0,05). Ova veća
HEV IgM seroprevalencija može se objasniti činjenicom da je riječ o pacijentima kod kojih je liječnik posumnjao na HEV
infekciju. Rezultati ove studije pokazuju da je, ukoliko pacijent ima simptome virusnog hepatitisa, a uobičajeni uzročnici
su isključeni (HAV, HBV, HCV, EBV, CMV), preporučljiva serološka dijagnostika na biljege virusnog hepatitisa E.
Statistički značajna razlika nije primijećena kod HEV IgG seroprevalencije (23,2 % naspram 20,2 %, χ2=1,77 <
χ2
df=1,α=0,05=3,84, p > 0,05) što upućuje na zaključak da je HEV i naša autohtona infekcija.The characteristic of hepatitis E virus (HEV) is that most people get over it asymptomatically. However, for certain
groups such as pregnant women, patients on immunosuppressive therapy and immunocompromised individuals with
pre-existing liver diseases, HEV infection can lead to a fatal outcome. During 16 months (2019 and 2020), slightly more
than 500 were serologically tested for hepatitis E markers at the Croatian Institute for Transfusion Medicine (CITM), in
the Department for Diagnostics of Blood Transmitted Diseases. Commercial HEV IgG Dia.Pro and HEV IgM Dia.Pro
tests were used. The principle of used method is ELISA. Tests were performed on Gemini analyzer and the complete
procedure followed the producer's instructions. Considering the results of patient testing (TP), the HEV IgG
seroprevalence is 23.2 % (117 samples) while the HEV IgM seroprevalence rate is 9.3 % (47 samples). In 2014, CITM
conducted a seroprevalence study that included 1036 samples from voluntary blood donors (VBD). HEV seroprevalence
in VBD was assessed with four available tests (3 ELISA and 1 immunoblot (IB) test). The results obtained with Dia.Pro
test were compared. HEV IgM seroprevalence among TP is significantly higher compared to VBD (9.3 % vs. 4.4 %) as
proven by the chi-square test (χ2 test) with a significance level of α=0.05 (χ2=15.13 > χ2
df=1,α=0.05=3.84, p < 0.05). This
higher HEV IgM seroprevalence can be explained by the fact that these are patients in whom the doctor suspected HEV
infection. The results of these studies show that, if the patient has symptoms of viral hepatitis, and the usual causative
agents are excluded (HAV, HBV, HCV, EBV, CMV), serological diagnosis for markers of viral hepatitis E is
recommended. No statistically significant difference was observed in HEV IgG seroprevalence (23.2 % vs. 20.2 %,
χ2=1.77 < χ2
df=1,α=0.05=3.84, p > 0.05) which leads to the conclusion that HEV is also our autochthonous infection
Kinetics and mechanism of reactions of homovanillyl alcohol and hydroxytyrosol with DPPH radical
No full textHidroksitirozol i homovanilinski alkohol smatraju se snažnim antioksidansima koji svojim antioksidativnim učincima ostvaruju niz biološki povoljnih učinaka koji se intenzivno proučavaju. Međutim, u literaturi nema veliki broj podataka o kinetičkim obilježjima i mehanizmu njihovih antioksidativnih reakcija. Prema strukturi molekule hidroksitirozol i homovanilinski alkohol razlikuju se u tome što hidroksitirozol u orto-položaju ima hidroksilnu skupinu, dok homovanilinski alkohol ima metoksi-skupinu, što uvelike utječe i na karakteristike njihovih reakcija. U ovom radu spektrofotometrijski su određeni kinetički parametri reakcija hidroksitirozola i homovanilinskog alkohola s DPPH● u otapalu 1,4-dioksan:voda = 99:1 (v/v). Određene su konstante brzine reakcija, Arrheniusovi i Eyringovi aktivacijski parametri, kinetički izotopni efekt te izotopne razlike aktivacijskih parametara. Konstanta brzine reakcije hidroksitirozola i DPPH● u iznosu od 5,46 mol-1 dm3 s-1 znatno je viša od konstante brzine reakcije homovanilinskog alkohola i DPPH koja iznosi 0,31 mol-1 dm3 s-1. Dobivena razlika u konstantama brzina reakcija upućuje na veću antioksidativnu aktivnost hidroksitirozola, što je s obzirom na razlike u strukturi i očekivano. Na veću antioksidativnu aktivnost hidroksitirozola upućuju i dobiveni iznosi Gibbsove aktivacijske energije od 69,00 kJ mol-1 za hidroksitirozol u odnosu na 82,49 kJ mol-1 za homovanilinski alkohol. Određen je i kinetički izotopni efekt (KIE) 16,88 za hidroksitirozol i 14,29 za homovanilinski alkohol. Navedeni iznosi KIE upućuju na efekt tuneliranja jer izlaze izvan granice maksimalnog mogućeg kinetičkog izotopnog efekta prema semiklasičnoj teoriji. Prisustvo KIE omogućuje eliminaciju uzastopnog elektron transfer/proton transfer (ET/PT) reakcijskog mehanizma. Koristeći dobivene iznose Gibbsove aktivacijske energije provedena je i termokemijska analiza (TKA). Na temelju provedene TKA eliminiran je i proton transfer/elektron transfer (PT/ET) mehanizam. Prema TKA, najvjerojatniji je mehanizam reakcije u kojemu dolazi do usklađenog prijelaza elektrona i protona (PCET). Dobiveni kinetički i termodinamički parametri mogu doprinijeti razumijevanju reakcija antioksidansa i fenola te pomoći rasvijetliti kinetičke i mehanističke karakteristike tih reakcija.Hydroxythyrosol and homovanillyl alcohol are considered to be strong antioxidants that, with their antioxidative effects, achieve a series of biologically favorable effects that are intensively studied. However, there is not a large amount of data in the literature on kinetic features and the mechanism of their antioxidant reactions. The molecular structure of these two molecules differs in that hydroxytyrosol has a hydroxyl group at the ortho-position, while at the same position homovanillyl alcohol has a methoxyl group. This difference also largely affects the characteristics of their reactions. In this thesis, the kinetic parameters of the reactions of the mentioned phenols were determined in a reaction with DPPH● in a reaction solvent mixture consisting of 1,4-dioxane:water = 99:1 (v/v). Reaction rate constants, Arrhenius and Eyring activation parameters, kinetic isotope effect, and isotopic differences of activation parameters were determined using the spectrophotometrical method. The reaction rate constant for reaction of hydroxytyrosol and DPPH● with the value of 5,46 mol-1 dm3 s-1 is significantly higher than the reaction rate constant of homovanillyl alcohol and DPPH●, which is 0,31 mol-1 dm3 s-1. Larger reaction rate constant for hydroxytyrosol reaction points to a higher antioxidant activity of hydroxytyrosol, which was also expected considering structural differences. The obtained values of Gibbs activation energy of 69,00 kJ mol-1 for hydroxytyrosol and 82,49 kJ mol-1 for homovanillyl alcohol also indicate the higher antioxidant activity of hydroxytyrosol. Both investigated phenols showed high values of kinetic isotope effect (KIE), 16,88 for hydroxytyrosol and 14,29 for homovanillyl alcohol. Both specified values of KIE point to the tunneling effect since they are outside the limit of the maximum possible kinetic isotope effect according to semiclassical theory. Moreover, the presence of KIE allows the elimination of the sequential electron transfer/proton transfer (ET/PT) as a possible reaction mechanism. Additionally, thermochemical analysis was also performed using the experimentally determined Gibbs activation energy.. Based on the performed thermochemical analysis and the comparison of the obtained Gibbs activation energies with the standard reaction Gibbs energies, the proton transfer/electron transfer (PT/ET) consecutive mechanism was also eliminated. According to the thermochemical analysis, the most probable reaction mechanism is proton-coupled electron transfer (PCET). Obtained kinetic and thermodynamic parameters can contribute to the understanding of the reactions of antioxidans and phenols and help us to ellucidate kinetic and mechanistic characteristics of these reactions
Left and right sided colon carcinoma - differences in pharmacotherapy
No full textKolorektalni karcinom jedan je od najučestalijih maligniteta u svijetu koji bilježi vrlo visoke stope morataliteta. Jedna od podjela kolorektalnog karcinoma je i ona prema položaju primarnog tumora prema kojoj se razlikuju lijevi i desni kolorektalni karcinom. Različit položaj karcinoma očituje se razlikama u procesu razvoja tumora i molekularnim karakteristikama što rezultira različitom kliničkom prezentacijom bolesti, ali i različitim odgovorom na farmakoterapiju. Iako nije zlatni standard, farmakoterapija je iznimno bitan dio liječenja kolorektalnog karcinoma. Osim što je izbor kod reduciranja tumorske mase prije operativnog zahvata, uvodi se i kada se želi smanjiti rizik ponovnog povratka bolesti, poboljšati kvalitetu života pacijenta ili kada je to jedina opcija liječenja. Farmakoterapija kolorektalnog karcinoma obuhvaća klasične kemoterapeutike, ciljanu terapiju te inhibitore imunosnih kontrolnih točaka. Budući da je kod pojedinih lijekova primijećen različit farmakoterapijski odgovor, izbor prikladne farmakoterapije jednim će dijelom ovisiti o tome radi li se o lijevom ili desnom kolorektalnom karcinomu. Farmakoterapija kod koje je primijećena najznačajnija razlika u odgovoru u ovisnosti o položaju karcinoma je ona usmjerena na EGFR i VEGF. Zbog navedenih razlika, tijekom postavljanja dijagnoze od velike je važnosti u obzir uzeti i položaj tumora kako bi se pacijentu osigurala optimalna terapija.Colorectal cancer is one of the most common malignancies worldwide and is associated with high mortality rates. One of the classifications of colorectal cancer is based on the location of the primary tumor, distinguishing between left-sided and right-sided colorectal cancer. The different positions of the cancer result in variations in tumor development processes and molecular characteristics, leading to distinct clinical presentations of the disease and different responses to pharmacotherapy. Although not the gold standard, pharmacotherapy plays a crucial role in the treatment of colorectal cancer. It is used to reduce tumor mass before surgical intervention, decrease the risk of disease recurrence, improve the patient's quality of life, or when it’s the only treatment option left. Pharmacotherapy for colorectal cancer includes conventional chemotherapeutics, targeted therapy, and immune checkpoint inhibitors. Since different pharmacotherapeutic responses have been observed with certain drugs, the choice of appropriate pharmacotherapy partially depends on whether it is left-sided or right-sided colorectal cancer. The pharmacotherapy that shows the most significant difference in response depending on the tumor location is directed towards EGFR and VEGF. Considering these differences, it is essential to take into account the tumor's position when establishing a diagnosis to ensure optimal therapy for the patient
Importance of immunohistochemical PD-L1 testing in treatment of urothelial carcinoma
No full textUrotelni karcinom (UC) čini 90% tumora bubrežne nakapnice, mokraćovoda i mokraćnog mjehura. Kod uznapredovale
bolesti u liječenju se primjenjuje kemoterapija, imunoterapija (inhibitori PD-1/PD-L1 imunosne kontrolne točke) i
potporno liječenje.
U ovom radu su prikazane karakteristike 97 pacijenata s UC kojima je u razdoblju od ožujka 2019. do kolovoza 2022.
u KBC-u Zagreb određivan PD-L1 status protutijelom SP142, obavezan za selekciju pacijenata pogodnih za primanje
atezolizumaba u prvoj liniji liječenja lokalno uznapredovalog ili metastatskog UC. Dobiveni rezultati su uspoređeni s
podacima iz drugih istraživanja.
Većinu pacijenata činili su muškarci (69; 71,13%). Pacijenti su bili dobi od 38 do 85 godina (medijan 70). Mokraćni
mjehur je bio najčešće primarno sijelo UC, nađen u 61 (62,89%) pacijenata. Zamijećen je znatno veći udio UC gornjeg
dijela mokraćnog sustava nego u literaturnim podacima: nakapnice bubrega u 22 (22,68%) pacijenta te mokraćovoda u
8 (8,25%) pacijenata. Kod 57 (58,76%) pacijenata UC je bio lokalno uznapredovao, a kod 36 (37,11%) su bile prisutne
udaljene metastaze. U 7 od 22 (31,82%) pacijenta s primarnim sijelom UC u nakapnici bubrega je nađen PD-L1 pozitivan
karcinom, dok je kod lokalizacije u mokraćovodu, mokraćnom mjehuru i kod multiplih sijela oko polovice karcinoma
PD-L1 pozitivno. Atezolizumab u prvoj liniji liječenja primilo je 6 (6,67%) pacijenata, a imunoterapiju u kasnijim
linijama liječenja 40 (44,44%) pacijenata (atezolizumab i nivolumab u omjeru 2,45:1). Ostali pacijenti nisu liječeni
imunoterapijom. U prvoj liniji, u skladu sa smjernicama, najčešće je primijenjeno liječenje kemoterapijskim protokolima
temeljenim na cisplatini (51 pacijenata; 56,67%) i karboplatini (12 pacijenata; 13,33%). Za 11 pacijenata nisu bile
dostupne informacije o ishodu. 7 (8,14%) pacijenata je stabilno bez progresije i bez dodatne terapije nakon primarnog
kirurškog liječenja; 30 (34,88%) pacijenata je stabilno na terapiji ili nakon nje, dok je kod 49 (56,98%) pacijenata
zabilježena progresija bolesti, smrtni ishod ili su izgubljeni iz praćenja u fazi značajne progresije.
Imunohistokemijsko određivanje PD-L1 statusa pokazalo se korisno za manji broj pacijenata kojima je omogućena
primjena imunoterapije u prvoj liniji liječenja, dok je vrijednost ove metode ograničena za pacijente koji su primali
imunoterapiju u kasnijim linijama liječenja.Urothelial carcinoma (UC) accounts for 90% of cancers in renal pelvis, ureter and bladder. Treatment options in
advanced disease include chemotherapy, immunotherapy (PD-1/ PD-L1 checkpoint inhibitors) and supportive care.
This study included 97 patients with locally advanced and metastatic UC tested with PD-L1 Ventana SP142 assay,
between 03/2019 and 08/2022 at University Hospital Centre Zagreb. The test was required for selection of patients with
PD-L1 positive tumors, eligible for the first-line treatment with atezolizumab. Obtained results were compared with data
from other studies.
The majority of patients were male (69; 71,13%). The age range was 38-85 years (median 70). Urinary bladder was the
most common primary site of UC, found in 61 (62,89%) patients, but UC originating from the upper urinary tract were
also more frequent than expected: renal pelvis (22; 22,68%) and ureter (8; 8,25%). 57 (58,76%) patients had locally
advanced carcinoma, while 36 (37,11%) had metastatic disease. PD-L1 positive UC was detected in 7 of 22 (31,82%)
cases originating from renal pelvis, while those arising from ureter, bladder and in multiple sites were PD-L1 positive
in roughly half of cases. 6 (6,67%) patients received atezolizumab as a first-line therapy, and 40 (44,44%) patients
received immunotherapy in a second or third-line setting (atezolizumab and nivolumab ratio 2,45:1). The rest of the
patients weren’t treated with immunotherapy. The majority of patients, in compliance with guidelines, received cisplatinbased (51; 56,67%) and carboplatin-based chemotherapy (12; 13,33%) as a first-line treatment. For 11 patients there
were no follow-up data. 7 (8,14%) patients after primary surgery show no progression without additional therapy; 30
(34,88%) patients are stable on/after therapy, while 49 (56,97%) patients experienced progression, were lost during
severe progression or died of disease.
Immunohistochemical determination of PD-L1 status proved to be useful for a selection of small number of patients
eligible for a first-line therapy with atezolizumab, while usefulness was limited for majority of patients who received
immunotherapy in a second or third-line settin
Virulence factors of the bacterium Enterococcus faecalis from different isolation sources
No full textBakterija Enterococcus faecalis je oportunistički patogen, poznata po komenzalističkom životu kao dio mikrobiote
različitih organskih sustava, a nalazi se i u okolišu. Uzrokuje infekcije urinarnog sustava, inficira kože i rane, a također
je specifična kao uzročnik endokarditisa. Sadrži mnoge virulentne čimbenike, a u ovom radu su detaljnije obrađeni
hidrofobnost stanične površine, sposobnost stvaranja biofilma, β-hemolitička aktivnost te osjetljivost na vankomicin.
Cilj diplomskog rada je bio utvrditi postoje li značajne razlike u uvjetima in vitro u virulenciji sojeva E. faecalis
izoliranih iz bolnice, hrane i otpadnih voda nakon statističke obrade rezultata. Istraživanjem hidrofobnosti stanične
površine zaključuje se da sojevi izolirani iz hrane imaju najveću hidrofobnost, dok između bolničkih izolata i onih iz
otpadnih voda nije bilo značajne razlike. Prema metodi iz rada Stępień-Pyśniak (2019), sposobnost stvaranja biofilma
je podijeljena u četiri kategorije te su sojevi iz bolničkog izvora najjači produktori biofilma, zatim sojevi iz hrane i
sojevi iz otpadnih voda kao najslabiji produktori biofilma. Ako se gleda sposobnost stvaranja biofilma samo prema
izmjerenoj optičkoj gustoći sojeva, najjači produktori biofilma su sojevi iz hrane potom iz otpadnih voda i konačno
bolnički izolati. Hemoliza je utvrđena u 20,00% sojeva iz bolničkog izvora, 16,67% iz hrane te 26,67% iz otpadnih
voda pri čemu Bonfferonijev test ukazuje da razlike između rezultata različitih sojeva nisu statistički značajne. Od
ukupno 30 sojeva iz svakog izvora, rezistenciju na vankomicin iz bolničkog izvora je pokazalo 10,00%, iz hrane 3,33%
i iz otpadnih voda 20,00%. Može se zaključiti da se nakon provedenih ispitivanja nulta hipoteza pokazala točnom kod
svih virulentnih čimbenika osim hemolize.The bacterium Enterococcus faecalis is an opportunistic pathogen known for its commensal life as part of the microbiota
of various organic systems and it can also be found in the environment. It causes infections of the urinary system, infects
skin and wounds, and is also a specific cause of endocarditis. It holds many virulent factors, of which the hydrophobicity
of the cell surface, the ability to form biofilm, β-hemolytic activity and susceptibility to vancomycin are discussed in
more detail in this paper. The aim of the thesis was to determine whether there are significant differences in in vitro
conditions the virulence of E. faecalis strains isolated from the hospital, food and wastewater after statistical data
processing. By investigating the hydrophobicity of the cell surface, it was concluded that the strains isolated from food
have the highest hydrophobicity, while there was no significant difference between hospital isolates and those from
wastewater. According to the method from the work of Stępień-Pyśniak (2019), the ability to form biofilm is divided
into four categories and those isolated from a hospital source are the strongest biofilm producers followed by strains
from food and strains from wastewater as the weakest biofilm producers. If the ability to form a biofilm is viewed only
according to the measured optical density of the strains, the strongest biofilm producers are strains isolated from food,
followed by isolates from wastewater and finally hospital isolates. Hemolysis was shown by 20,00% of strains from a
hospital source, 16,67% from food and 26,67% from wastewater, while the Bonfferoni test indicates that the differences
between the results of different strains are not statistically significant. Out of a total of thirty strains from each source,
10,00% showed resistance to vancomycin from a hospital source, 3,33% from food and 20,00% from wastewater. It can
be concluded that after the conducted tests, the null hypothesis proved to be correct for all virulence factors except
hemolytic activity
Valorization of tomato pomace as a source of nutritionally important minerals
No full textGodišnje u svijetu zaostane oko 8 milijuna tona otpada od prerade rajčice što ima negativne učinke na
okoliš te, s obzirom na činjenicu da je bogata esencijalnim nutrijentima i biološki vrijednim
fitokemikalijama predstavlja, veliki ekonomski gubitak.
Osnovni ciljevi ovog rada su istražiti primjenjivost rendgenske fluorescencijske spektrometrije s totalnom
refleksijom (TXRF) i zelenih tehnika pripreme uzorka u istraživanju mineralnog sadržaja različitih frakcija
komine rajčice sušenih u različitim uvjetima (sušenje u termostatu na 70 C i sušenje smrzavanjem).
Rezultati optimizacije metode ukazuju da kombinacija 1%-tnog Triton-a X-100®
uz izostavljanje koraka
soniciranja i volumen uzorka od 8 µL daje najtočnije rezultate.
Rezultati istraživanja su pokazali da se TXRF može koristiti u određivanju mineralnog sadržaja analiziranih
uzoraka te da priprema uzorka zelenim procesom suspendiranja daje jednako točne rezultate kao i
konvencionalne tehnike koje karakterizira puno veći ekološki otisak. Mineralni sadržaj komine rajčice u
skladu je s dostupnim literaturnim podacima, a način sušenja uzorka ne utječe značajno na mineralni sadržaj
analiziranih uzoraka. Rezultati ovog istraživanja doprinijeti će trenutnim saznanjima o prednostima i
nedostacima navedene analitičke tehnike te daljnjoj valorizaciji komine rajčice kao sekundarne sirovine
bogate nutritivno važnim mineralima.Annually, approximately 8 million tons of tomato processing waste is generated worldwide, which has a
negative environmental impact. Considering how rich in essential nutrients and biologically valuable
phytochemicals this waste is, this represents a significant economic loss.
The main objectives of this study are to investigate the applicability of total reflection X-ray fluorescence
spectrometry (TXRF) and green sample preparation techniques in exploring the mineral composition of
different fractions of tomato pomace dried under different conditions (thermostatic drying at 70°C and
freeze-drying). The results of the method optimization indicate that the combination of 1% Triton
X-100® with omission of sonication and sample volume of 8 µL gives most accurate results.
The results of this research have demonstrated that TXRF can be utilized for determining the mineral
composition of the analyzed samples. Furthermore, sample preparation using green suspension methods
yields equally accurate results as conventional techniques, characterized by a much larger ecological
footprint. The mineral composition of tomato pomace is in accordance with available literature data, and the
drying method does not significantly affect the mineral composition of the analyzed samples. The findings
of this research could contribute to current knowledge regarding the advantages and limitations of the
aforementioned analytical technique and further valorization of tomato pomace as a secondary resource rich
in nutritionally important minerals
Biodiversity of moulds on stored cereals
No full textKlimatske promjene postaju sve izraženije te ispoljavaju značajan učinak na rast plijesni i produkciju mikotoksina.
Porast temperature i koncentracije ugljikovog dioksida uz promjene količine padalina vode do promjene bioraznolikosti
mikrobioma te veće kontaminacije prehrambenih namirnica. Kolonizirani usjevi predstavljaju veliku štetu
agrikulturalnom sektoru, ali i prijetnju ljudskom zdravlju.
Analizirano je 90 uzoraka žitarica iz skladišta u ožujku 2022., prikupljenih na području sjeverne, središnje i istočne
Hrvatske. Istraživanje je pokazalo najveću koncentraciju plijesni na uzorcima kukuruza iz sjeverne Hrvatske na obje
hranjive podloge (2,8 x 106 CFU/g na DG-18 agaru te 5,3 x 106 CFU/g na DRBC agaru).
Superioran rast pokazale su plijesni roda Penicillium s prosječnom zastupljenošću iznad 50%. Iduće po učestalosti bile
su plijesni roda Fusarium čiji je rast dominirao na uzorcima kukuruza. Porast plijesni roda Cladosporium, kao i kvasaca
također je u većoj mjeri bio prisutan. Iako se aflatoksini smatraju najvećom prijetnjom ljudskom zdravlju, u provedenom
istraživanju rast je plijesni roda Aspergillus sekcije Flavi zabilježen s maksimalnom učestalošću od tek 30% na uzorcima
kukuruza iz istočne Hrvatske (DG-18). Narušenost kvalitete žitarica najviše je vidljiva na uzorcima kukuruza koji se
smatraju čak neprihvatljivim za hranidbu životinja.
Briga o ispravnom sušenju te skladištenju žitarica, kao i spriječavanje nastanka rana od strane insekata, kritične su točke
životnog ciklusa žitarica. Zadržavanje kvalitete i sigurnosti žitarica može se postići osiguravanjem dobre poljoprivredne
prakse (GAP) te pomoću sustava za očuvanje sigurnosti hrane (HAACP).
Utjecaj na industrijsku emisiju plinova može biti od ključne važnosti za prevenciju rasta plijesni te očuvanje ljudskog zdravlja,
posebice uzimajući u obzir promjene koje se očekuju na razini klimatskih uvjeta.Climate changes are becoming more pronounced and have a significant effect on mold growth and mycotoxin
production. An increase in temperature and concentration of carbon dioxide along with changes in the amount of rainfall
lead to a change in the biodiversity of the microbiome and greater contamination of foodstuffs. Colonized crops represent
a great damage to the agricultural sector, but also a threat to human health.
90 grain samples from warehouses in March 2022, collected in northern, central and eastern Croatia, were analyzed.
The research showed the highest concentration of mold on corn samples from northern Croatia on both used media (2.8
x 106 CFU/g on DG-18 agar and 5.3 x 106 CFU/g on DRBC agar).
Superior growth was shown by the mold genera Penicillium with an average frequency above 50%. Next in order of
frequency were molds of the genera Fusarium, whose growth dominated on the corn samples. The growth of molds of
the genera Cladosporium, as well as yeasts, was also present to a greater extent. Although aflatoxins are considered the
greatest threat to human health, in the present study, the growth of Aspergilli section Flavi was recorded with a maximum
frequency of only 30% on corn samples from eastern Croatia (DG-18). The impairment of grain quality is most visible
on corn samples that are considered even unacceptable for feeding animals.
Taking care of the drying and storage conditions as well as the prevention of wounding by insects, are critical points in
the life cycle of grains. Maintaining the quality and safety of grains can be achieved by ensuring Good Agricultural
Practices (GAP) and using the Hazard Analysis and Critical Control Point (HAACP).
The impact on industrial gas emissions can be of crucial importance for the prevention of mold growth and the
preservation of human health, especially taking into account the expected changes in climate conditions
The impact of the chromogenic method for determining the activity of coagulation factor IX on the classification and treatment of patients with hemophilia B
No full textHemofilija B je rijetki X-vezani nasljedni poremećaj zgrušavanja krvi uzrokovan potpunim ili djelomičnim
manjkom aktivnosti koagulacijskog faktora IX što rezultira povećanom sklonosti krvarenjima. S obzirom kako u
većini slučajeva ostatna aktivnost FIX dobro korelira s kliničkom slikom, pacijenti s hemofilijom B klasificiraju
se kao teški, umjereni ili blagi oblik na temelju aktivnosti FIX izmjerenih koagulacijskom metodom. S obzirom
na sve veću primjenu kromogene metode u dijagnostici hemofilije i njezinih prednosti u odnosu na koagulacijsku
metodu, ovim se istraživanjem nastojalo ispitati kakav utjecaj određivanje aktivnosti FIX kromogenom metodom
ima na klasifikaciju pacijenata s hemofilijom B i ispitati razlike u rezultatima terapijskog praćenja pacijenata
koagulacijskom i kromogenom metodom kako bi se utvrdilo koja je metoda prikladnija za praćenje učinkovitosti
lijekova te time doprinijelo boljoj skrbi i liječenju pacijenata s hemofilijom B. U istraživanje je bilo uključeno 45
ispitanika muškog spola (33 odrasla i 12 pedijatrijskih) koji boluju od hemofilije B, od kojih je pet u trenutku
ispitivanja primalo nadomjesnu terapiju te su oni promatrani kao zasebna skupina prilikom statističke obrade
rezultata. Svim je ispitanicima izmjereno APTV te aktivnosti FIX koagulacijskom metodom (FIXkgl) i
kromogenom metodom (FIXkr) upotrebom BIOPHENTM FIX komercijalnog testa (HYPHEN BioMed, Neuville
sur Oise, Francuska) na analizatoru Atellica COAG 360 (Siemens Healthcare, Marburg, Njemačka). U odnosu na
aktivnosti FIXkr, aktivnosti FIXkgl su bile značajno više u pacijenata s blagim i umjerenim oblikom hemofilije B
uz iznimku teškog oblika gdje su primijećene više vrijednosti FIXkr. Najviše je odstupanja u klasifikaciji s obje
metode uočeno kod teškog i umjerenog oblika hemofilije B dok je najmanje odstupanja u klasifikaciji zabilježeno
kod blagog oblika hemofilije. Također je uočena bolja povezanost kliničkog fenotipa s klasifikacijom prema
kromogenoj metodi. U skupini pacijenata na nadomjesnoj terapiji statističkom analizom nije uočena statistički
značajna razlika aktivnosti FIXkr odnosu na aktivnosti FIXkgl. Najveća odstupanja između rezultata FIXkr i
FIXkgl u skupini pacijenata na nadomjesnoj terapiji uočena su u području viših aktivnosti FIX. Rezultati ovog
istraživanja ukazali su na složenost diferencijalno-dijagnostičkog pristupa u dijagnostici hemofilije B, kao i na
neizostavnu ulogu koagulacijske i kromogene metode u dijagnostici i praćenju nadomjesne terapije. Nadalje,
uočene su prednosti i nedostaci pojedine metode te je osigurana osnova za potencijalnu primjenu kromogene
metode u klasifikaciji i terapijskom praćenju pacijenata s hemofilijom B.Hemophilia B is a rare X-linked hereditary blood clotting disorder caused by a complete or partialy deficiency of
coagulation factor IX activity that results with increased bleeding tendency. Since residual FIX activity correlates
well with the clinical picture in most cases, hemophilia B patients are classified as severe, moderate or mild based
on FIX activity measured by the one-stage coagulation method. With regard to the increasing use of the
chromogenic method in the diagnosis of hemophilia and its advantages compared to the standard coagulation
method, the goal of this investigation was to examine the influence of the determination of FIX activity by the
chromogenic method on the classification of hemophilia B patients and to examine the differences in the results
of the therapeutic monitoring by coagulation and chromogenic methods in order to determine which method is
more suitable for monitoring the effectiveness of drugs and thereby contributes to better care and treatment of
hemophilia B patients. The investigation has included 45 male subjects (33 adults and 12 pediatric) suffering from
hemophilia B, of whom five were receiving replacement therapy at the time of the examination, and they were
obsereved as a separate group during the statistical analysis of results. APTT and FIX activities were measured by
using the coagulation method (FIXkgl) and chromogenic method (FIXkr) using the BIOPHENTM FIX commercial
test (HYPHEN BioMed, Neuville sur Oise, France) in all subjects on the Atellica COAG 360 analyzer (Siemens
Healthcare, Marburg, Germany). In relation to FIXkr activities, FIXkgl activities were significantly higher in mild
and moderate hemophilia B patients with the exception of the severe form where higher FIXkr values were
observed. The most discrepancies in classification with both methods were observed in severe and moderate
hemophilia B forms, while the least discrepancies in classification were observed in mild forms of hemophilia.
Moreover, a better correlation of the clinical phenotype with the classification according to the chromogenic
method was observed. In the group of patients on replacement therapy, statistically significant difference was not
found between the activities of FIXkr compared to FIXkgl activities. The largest discrepancies between the results
of FIXkr and FIXkgl in the group of patients on replacement therapy were observed in the area of higher FIX
activities. The results of this investigation indicated the complexity of the differential-diagnostic approach in the
diagnosis of hemophilia B and the indispensable role of the coagulation and chromogenic methods in the diagnosis
and monitoring of replacement therapy. Also, the advantages and disadvantages of each method were observed,
and the basis for the potential application of the chromogenic method in the classification and therapeutic
monitoring of patients with hemophilia B was provided