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Utjecaj preferencijalne konzumacije metamfetamina na dužinu životnog ciklusa i bihevioralne fenotipove Drosophile melanogaster
No full textAddiction is a relapsing disease caused by substance abuse. Methamphetamine and cocaine are potent psychostimulants whose abuse induces numerous negative consequences for the abuser. Studying substance abuse in humans is challenging, however its effects can be analyzed in model organisms such as Drosophila melanogaster. Alongside their genetic traceability, fruit flies are extensively researched making them exemplary models for addiction research. Methamphetamine and cocaine disbalance monoaminergic levels, damage axons and create reactive oxygen species which disrupt normal organism functions. Considering that addiction shows around 72% heritability, we were wondering if the effects of paternal methamphetamine abuse can be seen in filial generations. After artificial selection of the 28th Canton S fly generation with high (HP) and low (LP) preference for methamphetamine, we observed life cycle and behavioral phenotypes of flies, their morphology and voluntary self-administration of methamphetamine through FlyCAFE assay. Our aim was to determine if there are any differences between wt, HP and LP lines after 28 generations of selection. The obtained data shows impaired activity, sleep, and negative geotaxis of flies, alongside possible changes in flies weight and size compared to control wt line. Locomotor activity of HP, LP, and wt flies increases after volatilized methamphetamine and cocaine administration with various significances among lines. The HP and LP flies maintain the same preference for methamphetamine, high and low, respectively. Finally, there is no difference in length of life cycle among lines, but HP flies have lower reproduction success rate. Our findings point to a transgenerational effect of paternal methamphetamine abuse, posing further questions about the epigenetic and/or genetic changes induced by substance abuse.Ovisnost je relapsirajuća bolest uzrokovana zlouporabom opojnih supstanci. Metamfetamin i kokain su snažni psihostimulansi čija konzumacija ostavlja brojne negativne posljedice na organizam ovisnika. Izučavanje zlouporabe doga u ljudi vrlo je izazovno, stoga se njihovi učinci analiziraju u modelnim organizmima poput vinske mušice (Drosophila melanogaster). Vinske mušice pogodne su za istraživanja ovisnosti jer imaju dobro proučen i sekvenciran genom pa se njihove genetske promjene mogu jednostavnije pratiti i izučavati. Metamfetamin i kokain uzrokuju poremećaje u nivoima monoamina, oštećuju aksone te stvaraju reaktivne kisikove vrste, što otežava razne funkcije organizma. S obzirom na činjenicu da ovisnost pokazuje oko 72% nasljednosti, interesiralo nas je da li se učinci paternalne preferencijalne samo-administracije metamfetamina očituju u filijalnim generacijama mušica. Nakon umjetne selekcije 28. generacije mušica Canton S linije sa visokom (HP) i niskom (LP) preferencijom za metamfetamin, proučavali smo životni ciklus i bihevioralne fenotipove D. melanogaster. Također smo promatrali morfologiju mušica i larvi te samo-administraciju metamfetamina kroz FlyCAFE metodu. Cilj ove teze bio je vidjeti postoje li razlike između wt, HP i LP linija nakon 28 generacija umjetne selekcije. Prikupljeni rezultati ukazuju na povećanu aktivnost i smanjen san mušica, kao i na smanjenu sposobnost negativne geotaksije. Nadalje, postoji mogućnost da visoka i niska paternalna preferencijalna samo-administracija metamfetamina utječe na veličinu i težinu mušica, odnosno larvi u odnosnu na wt. Lokomotorna aktivnost mušica HP, LP i wt linija raste nakon administracije volatiliziranog metamfetamina i kokaina sa raznim nivoima statističke značajnosti unutar linija. HP linija zadržava visoku, a LP linija nisku preferenciju metamfetamina, što je u skladu s prethodno testiranim generacijama. Konačno, nema promjena u duljini životnog ciklusa mušica, no HP linija ima nižu reprodukcijsku uspješnost. Navedeni rezultati ukazuju na trans-generacijski efekt paternalne preferencijalne samo-administracije metamfetamina, te postavljaju pitanja o potencijalnim epigenetskim ili genetskim promjenama koje su inducirane zlouporabom supstanci
Utjecaj ekstrakata nefermentiranog i fermentiranog japanskog dvornika (Fallopia japonica) na stupanj oksidacije u kvascu Saccharomyces cerevisiae
No full textThe process of oxidation within the human body causes damage to various cellular components. During this process, free radicals are created which cause cellular damage. Antioxidants are compounds that prevent damage within cells by neutralizing the free radicals or by interfering in chain reactions involving free radicals. Despite the reputation of Japanese knotweed (Fallopia japonica) as the most invasive species, it has been shown to be a high source of different bioactive compounds with potential antioxidant activity. Lactic acid fermentation can be used to improve the nutritional as well as functional value of substrates including antioxidant activity. This study aimed to compare the impact of non-fermented and fermented Japanese knotweed on oxidation level in yeast Saccharomyces cerevisiae. The fermentation of F. japonica was carried out by Lactobacillus plantarum and evaluated by measuring pH values and L. plantarum (LAB) growth. In ethanol extracts prepared from fermented and non-fermented F. japonica, total phenolic content using the Folin- Ciocalteu reagent, antioxidant activity by DPPH assay, and antioxidant activity in the cells were measured. The maximum decrease in pH value is observed in the first 24 hours. However, the maximum LAB growth is seen in the first 48 hours. Total phenolic content decreased in 48 hours of fermentation, but DPPH radical scavenging activity increased in the first 48 hours of fermentation. The antioxidant capability of F. japonica was further proved with the decrease of oxidation level in cells of yeast Saccharomyces cerevisiae. Therefore, the biomass of F. japonica has demonstrated its potential as a source of antioxidants.Proces oksidacije uzrokuje oštećenje različitih staničnih komponenti unutar ljudskog tijela. Tijekom tog procesa stvaraju se slobodno radikali koji uzrokuju oštećenje stanica. Antioksidansi su spojevi koji sprječavaju oštećenja unutar stanica neutraliziranjem slobodnih radikala ili miješanjem u lančane reakcije koje uključuju slobodne radikale. Unatoč reputaciji japanskog dvornika (Fallopia japonica) kao najinvazivnije vrste, pokazalo se da je visoki izvor različitih bioaktivnih spojeva s potencijalnim antioksidativnim djelovanjem. Mliječna fermentacija može se koristiti za poboljšanje nutritivne, kao i funkcionalne vrijednosti supstrata, uključujući antioksidativno djelovanje. Cilj ovog istraživanja je usporediti utjecaj nefermentiranog i fermentiranog japanskog dvornika na razinu oksidacije u kvascu Saccharomyces cerevisiae. Fermentacija F. japonica provedena je pomoću Lactobacillus plantarum i određena mjerenjem pH vrijednosti i rasta L. plantarum (LAB). U etanolnim ekstraktima pripremljenim iz fermentirane i nefermentirane F. japonica, izmjeren je ukupni sadržaj fenola korištenjem Folin-Ciocalteu reagensa, antioksidacijska aktivnost DPPH testom i antioksidativna aktivnost u stanicama. Maksimalno smanjenje pH vrijednosti uočeno je u prvih 24 sata. Međutim, najveći rast LAB-a vidljiv je u prvih 48 sati. Ukupni sadržaj fenola smanjio se u 48 sati fermentacije, ali se aktivnost hvatanja DPPH radikala povećala u prvih 48 sati fermentacije. Antioksidativna sposobnost F. japonica dodatno je dokazana smanjenjem razine oksidacije u stanicama kvasca Saccharomyces cerevisiae. Stoga je biomasa F. japonica pokazala svoj potencijal kao izvor antioksidansa
Serološka dijagnostika COVID-19: usporedba imunoenzimskog testa, surogat neutralizacijskog testa i testa neutralizacije virusa
No full textThe sensitivity and specificity of SARS-CoV-2 serological tests vary widely. The results of three serological tests: EIA (binding antibodies), VNT (NT antibodies), and FIA (sVNT; sNT antibodies) were compared in three groups of patients: vaccinated individuals (N=62); patients who recovered from COVID-19 (N=62) and vaccinated individuals infected with SARS-CoV-2 after vaccination (N=28). SARS-CoV-2 binding antibodies, NT antibodies, and sNT antibodies were detected in 93.1%, 84.2%, and 85.6% of patients. The highest detection rates were observed in vaccinated + COVID-19 patients (100/96.4/100%), compared to 100/96.7/85.5% in COVID-19 patients. The lowest detection rates were in vaccinated individuals (88.5/69.2/84.6%). In
There were significant differences in antibody levels between groups tested using EIA. NT antibody titers did not differ among vaccinated individuals and COVID-19 patients; however, in vaccinated + COVID-19 patients, significant differences were found using different viral strains. The lowest median titer was for the Omicron variant (64) compared to the Alpha variant (256), Delta variant (128), and Wuhan strain (128). Additionally, the sNT antibody levels differed between groups with the highest positivity of inhibition (median) in vaccinated + COVID-19 patients (100%) in comparison with COVID-19 patients (92%) and vaccinated individuals (96%). In vaccinated individuals, there was a significant strong positive correlation between EIA/VNT, EIA/sVNT, and VNT/sVNT. COVID-19 patients showed a moderate positive correlation between all serological tests, while vaccinated + COVID-19 patients showed a significant moderate positive correlation only between EIA and sVNT. The frequency of positive sVNT detection rates depended on the NT titer and was lowest in samples with low NT titers (8 and 16). The presented results showed that sVNT is useful in patients with high antibody levels. Due to the possibility of false-negative results, it cannot entirely replace the traditional VNT.Osjetljivost i specifičnost seroloških testova za SARS-CoV-2 uvelike varira. Rezultati triju seroloških testova: EIA (IgG protutijela), VNT (NT protutijela) i FIA (sVNT; sNT protutijela) uspoređeni su u tri skupine bolesnika: cijepljene osobe (N=62); bolesnici koji su preboljeli COVID-19 (N=62) i cijepljene osobe zaražene sa SARS-CoV-2 nakon cijepljenja (N=28). SARS-CoV-2 protutijela koja se dokazuju u EIA, NT protutijela i sNT protutijela otkrivena su u 93,1%, 84,2% i 85,6% bolesnikaa. Najviše stope detekcije opažene su kod cijepljenih + COVID-19 bolesnika (100/96,4/100%), u usporedbi sa 100/96,7/85,5% kod bolesnika s COVID-19. Najniže stope detekcije bile su u cijepljenih osoba (88,5/69,2/84,6%). Uočene su značajne razlike u razinama protutijela između skupina testiranih metodom EIA. Titrovi NT protutijela nisu se razlikovali među cijepljenim osobama i bolesnicima s COVID-19; međutim, u cijepljenih + COVID-19 bolesnika, značajne razlike nađene pri testiranju s različitim sojevima virusa. Najniži medijan titra bio je za varijantu Omicron (64) u usporedbi s varijantom Alpha (256), varijantom Delta (128) i Wuhanskim sojem (128). Osim toga, razine sNT protutijela razlikovale su se između skupina s najvišim postotkom inhibicije (medijan) u cijepljenih + COVID-19 bolesnika (100%) u usporedbi s COVID-19 bolesnicima (92%) i cijepljenim osobama (96%). Kod cijepljenih osoba postojala je značajna jako pozitivna korelacija između EIA/VNT, EIA/sVNT i VNT/sVNT. Bolesnici s COVID-19 pokazali su umjerenu pozitivnu korelaciju između svih seroloških testova, dok su cijepljeni + COVID-19 bolesnici pokazali značajnu umjerenu pozitivnu korelaciju samo između EIA i sVNT. Učestalost pozitivnih stopa detekcije sVNT ovisila je o NT titru i bila je najniža u uzorcima s niskim NT titrovima (8 i 16). Prikazani rezultati pokazali su da je sVNT koristan kod bolesnika s visokim razinama protutijela, no zbog mogućnosti lažno negativnih rezultata, ne može u potpunosti zamijeniti klasični VNT
Plan upravljanja istraživačkim podacima - IP-2020-02-2287
No full textPlan upravljanja istraživačkim podacim
Immunogenicity of medicinal products containing monoclonal antibodies
No full textLijekovi koji sadrže monoklonska protutijela su visoko specifični te se
poglavito u proteklih desetak godina, koriste u svrhe dijagnostike i terapije
autoimunih bolesti te raka. Primjena monoklonskih protutijela može
uzrokovati reakcije preosjetljivosti. Takve reakcije najčešće se očituju na
koži i općenito imaju povoljne ishode. Ipak, potrebno je sustavno praćenje
reakcija preosjetljivosti kako bi se optimizirao omjer koristi i štetnosti
njihove primjene. Bazu podataka o svim nuspojavama, pa tako i reakcijama
preosjetljivosti, vodi Hrvatska agencija za lijekove i medicinske proizvode
(HALMED). U ovom diplomskom radu analizirane su prijavljene reakcije
preosjetljivosti na lijekove koje sadrže monoklonska protutijela prijavljenih
HALMED-u. Prijave nuspojava analizirane su po godini zaprimanja,
prijavitelju, ozbiljnosti, kriteriju ozbiljnosti, dobi, spolu, preferiranom
pojmu (eng. Preferred term) kao i samoj djelatnoj tvari. HALMED je do 15.
svibnja 2023. godine zaprimio 3859 prijava nuspojava na monoklonska
protutijela, od kojih je 62,3% bilo ozbiljno. 796 prijava iz navedenog
razdoblja (20,6% svih prijava) odnosilo se na reakcije preosjetljivosti na
monoklonska protutijela. Najčešći prijavitelji reakcija preosjetljivosti bili su
liječnici (86,3%). Najviše prijava reakcija preosjetljivosti je zabilježeno kod
ženske populacije (49,1%) i kod odraslih pacijenata u dobi od 45 do 64
godina (29,8%). Više od polovice (54,1%) prijavljenih slučajeva reakcija
preosjetljivosti nije bio ozbiljan. Najčešće prijavljene nuspojave koje
spadaju u reakcije preosjetljivosti bile su osip, eritem i svrbež. Najveći broj
reakcija preosjetljivosti prijavljen je za sljedeće djelatne tvari: infliksimab,
pembrolizumab i adalimumab. Prema rezultatima ovog diplomskog rada
reakcije preosjetljivosti su često prijavljivane nuspojave dok u usporedbi s
drugim nuspojavama, manji udio nuspojava je ocijenjen ozbiljnim. Ovi
rezultati naglašavaju važnost sveobuhvatnog praćenja i evaluacije reakcija
preosjetljivosti na lijekove koji sadrže monoklonska protutijela kako bi se
osigurala njihova sigurna i učinkovita uporaba u kliničkoj praksi.Medicinal products containing monoclonal antibodies are highly specific and
they have been used mainly for the diagnosis and therapy of autoimmune
diseases and cancer for the past ten years. However, the use of monoclonal
antibodies can cause hypersensitivity reactions. Typically, these reaction
manifest on the skin and generally have favorable outcomes. Nevertheless,
systematic monitoring of hypersensitivity reactions is necessary in order to
optimize the benefit-harm ratio of their use. The Croatian Agency for
Medicines and Medical Products (HALMED) maintains a comprehensive
database encompassing all adverse effects, including hypersensitivity
reactions. This thesis analyzes cases of reported hypersensitivity reactions
to monoclonal antibody-based medications reported to HALMED. Adverse
reaction reports were analyzed by year of receipt, reporter, seriousness,
seriousness criteria, age and gender of a patient, preferred term, as well
as the active substance. By May 15, 2023, HALMED had received 3,859
reports of adverse reactions to monoclonal antibodies, with 62.3%
classified as serious. Of these reports, 796 (equivalent to 20.6% of all
reports) corresponded to hypersensitivity reactions to monoclonal
antibodies. Medical professionals constituted the primary reporters of
hypersensitivity reactions, accounting for 86.3% of the reports.
Hypersensitivity reactions were more frequently reported among female
patients (49.1%) and adults aged 45 to 64 (29.8%). A majority of the
documented hypersensitivity cases were categorized as non-serious
(54.1%). Commonly reported symptoms included rash, erythema, and
pruritus. The active substances most frequently associated with
hypersensitivity reactions were infliximab, pembrolizumab, and
adalimumab. According to the results of this thesis, hypersensitivity
reactions are frequently reported side effects, while compared to other
reported side effects, a smaller proportion of reported side effects are
assessed as serious. These results highlight the importance of
comprehensive monitoring and evaluation of hypersensitivity reactions to
medicinal products containing monoclonal antibodies to ensure their safe
and effective utillization in clinical practice
Razvoj LC-MS/MS metode u svrhu ispitivanja utjecaja ozljede leđne moždine na disbiozu i količinu kratkolančanih masnih kiselina u štakorima
No full textSpinal cord injury (SCI) has shown to induce changes in healthy gut
microbiome and cause gut dysbiosis, leading to a reduction of bacteria
responsible for the synthesis of short-chain fatty acids (SCFA). SCFAs have
a variety of anti-inflammatory and immune properties, and it has been
hypothesised that SCI leads to changes in concentration of circulating
SCFAs, leading to a variety of health issues, including neural damage. Due
to this, SCFAs pose as clinical markers, and the effect of SCI on SCFA
concentration can be studied in rat models. SCFAs are usually quantified
using gas chromatography coupled with mass spectrometry (GC-MS)
because of their volatility. However, there is a growing number of
quantification studies done using liquid chromatography coupled with mass
spectrometry (LC-MS). To examine SCFAs as clinical markers, the
derivatization and extraction methods were optimised as were parameters
used for multiple reaction monitoring (MRM) using tandem mass
spectrometry. The protocol included derivatisation using 3-
nitrophenylhydrazine hydrochloride (3-NPH). Following derivatisation,
eleven SCFA standards were injected onto a liquid chromatography tandem
mass spectrometry (LC-MS/MS) system for MRM parameters optimization.
Deuterated internal standards (ISTD) were used to identify and account for
the matrix effect (ME) and method recovery. SCFAs from plasma samples
of SCI rats and sham-control rats (n=5 per group) were extracted and
analysed using the LC-MS/MS method to test whether there is a difference
in SCFA concentration between sham and SCI samples. Results showed a
successful chromatographic separation of all SCFA standards, however only
three out of eleven SCFAs showed a statistically significant difference in
concentrations between SCI and sham groups. To draw firm conclusions,
the experiment should be performed on a larger sample size.Istraživanja su pokazala kako ozljeda leđne moždine uzrokuje promjene u
crijevnoj mikrobioti. Takve promjene uzrokuju disbiozu i smanjenje broja
bakterija zaslužnih za sintezu kratkolančanih masnih kiselina.
Kratkolančane masne kiseline imaju protuupalna i imunomodulatorna
svojstva, te se smatra kako ozljeda leđne moždine uzrokuje promjene
njihove koncentracije u cirkulaciji i uzrokuje negativne učinke na zdravlje.
Zahvaljujući tome, kratkolančane masne kiseline su potencijalni klinički
markeri za ispitivanje raznih bolesti, a najprikladniji model za ispitivanja su
štakori. Zbog svojstva hlapljivosti, kratkolančane masne kiseline obično se
kvanitificiraju koristeći plinsku kromatografiju spregnutu sa
spektrometrijom masa (GC-MS), no sve veći broj ispitivanja koristi
tekućinsku kromatografiju spregnutu sa spektrometrijom masa (LC-MS).
Da bi se kratkolančane masne kiseline ispitivale pomoću LC-MS metode,
metode derivatizacije i ekstrakcije moraju biti optimizirane. Protokol u ovom
radu uključuje derivatizaciju pomoću 3-nitrofenilhidrazin hidrokorida.
Nakon procesa derivatizacije, jedanaest standarada kratkolančanih masnih
kiselina se injektiralo u LC-MS/MS te su optimizirani parametri za
kvantifikaciju. Deuterirani interni standardi su korišteni za supresiju efekta
matriksa i za određivanje oporavka metode. Uzorci plazme za kvantifikaciju
kratkolančanih masnih kiselina prikupljeni su iz pet štakora s induciranom
ozljedom leđne moždine (SCI), te pet štakora sa zdravom leđnom
moždinom (SHAM). Rezultati su pokazali uspješno kromatografsko
razdvajanje kratkolančanih masnih kiselina, no samo tri od jedanaest
kratkolančanih masnih kiselina je pokazalo statistički značajnu razlika
između SCI i SHAM grupe. Zbog malog broja uzoraka, ovaj se eksperiment
mora ponoviti na većem broju kako bi se mogli donesti konačni zaključci
Razlike u rijetkim kodonima utječu na ekspresiju neurorazvojnih proteinskih paraloga NDE1 i NDEL1
No full textChronic mental illnesses are persistent and disrupt cognition, emotion regulation and behaviour, and their exact causes and mechanisms remain largely unknown. Previous studies identified several genetic factors that play a role in their development. Nuclear Distribution Element 1 (NDE1) and Nuclear Distribution Element-Like 1 (NDEL1) are proteins that arise form a gene duplication event and are vital in cell mitosis and neurodevelopment. They have been associated with brain malformations and neurodevelopmental disorders. Although being structurally similar, they exhibit distinct pathophysiological functions.
Rare codon bias is a tendency for some codons to be more frequently used than others in a specific species, while they encode for the same amino acid. Codon rarity has been demonstrated to affect translation speed, protein folding, translational control, and protein expression levels in multiple known genomes. In human NDE1 and NDEL1 genes, the latter shows an increase in frequency of rare codon usage.
In this thesis, I first investigated whether the difference in codon rarity between NDE1 and NDEL1 was conserved across multiple species that represented major vertebrate genera. Next, I explored if altering the codons responsible for encoding NDE1 and NDEL1 could mitigate the differences in expression levels between these two proteins. To achieve this, I examined NDE1 constructs that used codons closely, matching ones found in NDEL1, while maintaining the correct amino acid sequence, and vice versa. Finally, I examined the expression patterns of both wild type and switched codon NDE1 and NDEL1 proteins by fluorescent microscopy with the aim of identifying any noticeable distinctions if such differences existed.
The preference of NDEL1 for rare codon usage that had previously been seen in humans, was also seen in non-human primates and non-primate mammals, suggesting the rare codon bias differences between the two genes to be conserved across mammalian species. Our findings indicated that the difference in codon rarity in wild type NDE1 and NDEL1 had an impact on their respective expression levels, with NDE1 exhibiting higher expression within cells. Notably, when examining constructs with switched codons, there was indication of this difference being nullified. This observation suggests that the use of more commonly occurring codons could potentially reverse the trend of lower protein expression levels and that rare codon bias may be partially responsible for the differing functions of the two proteins.Kronične mentalne bolesti su dugotrajne i ometaju kogniciju, regulaciju emocija i ponašanje, a njihovi točni uzroci i mehanizmi uglavnom su nepoznati. Prethodne studije identificirale su nekoliko gena koji igraju ulogu u njihovom razvoju. Nuklearni distribucijski element 1 (NDE1) i nuklearni distribucijski element sličan 1 (NDEL1) su proteini nastali duplikacijom gena koji sudjeluju u staničnoj mitozi i neurološkom razvoju te su povezani s malformacijama mozga i neurološkim poremećajima. Iako su strukturno slični, njihove se patofiziološke funkcije značajno razlikuju.
Pristranost rijetkim kodonima je tendencija da se neki kodon koristi češće od drugih u određenoj vrsti, kada je riječ o onima koji kodiraju istu aminokiselinu. Kod ljudi, NDEL1 gen pokazuje veću učestalost korištenja rijetkih kodona u odnosu na NDE1 gen. Pokazalo se da rijetkost kodona utječe na brzinu translacije, savijanje proteina, kontrolu translacije i razine ekspresije proteina u većem broju istraženih genoma.
U diplomskom radu najprije sam istražila je li razlika u rijetkosti kodona između NDE1 i NDEL1 očuvana u više reprezentativnih vrsta rodova kralješnjaka. Zatim sam istražila može li zamjena kodona odgovornih za kodiranje NDE1 i NDEL1 ublažiti razlike u razinama ekspresije između ova dva proteina. U tu svrhu ispitivala sam NDE1 konstrukte kodirane kodonima bliskim onima koje koristi NDEL1 uz očuvanje točne aminokiselinske sekvence, i obrnuto. Na kraju sam fluorescentnom mikroskopijom ispitala gdje se unutar stanica eksprimiraju NDE1 i NDEL1 proteini divljeg tipa i onih s promijenjenim kodonima kako bi utvrdili postoje li očite razlike.
Pristranost za rijetke kodone kod NDEL1 ranije je uočena kod ljudi, a sada i kod primata i sisavaca koji nisu primati, što ukazuje da je razlika u pristranosti evolucijski očuvana u sisavcima. Naši rezultati pokazali su da je razlika u rijetkosti kodona u divljem tipu NDE1 i NDEL1 imala utjecaj na njihovu razinu ekspresije, pri čemu NDE1 pokazuje veću ekspresiju unutar stanica. Pri ispitivanju konstrukata sa zamijenjenim kodonima, postojala je indikacija da je ova razlika u ekspresiji poništena. Ovo opažanje sugerira da bi korištenje kodona koji se češće pojavljuju moglo potencijalno preokrenuti trend nižih razina ekspresije proteina, te da bi pristranost za rijetke kodone mogla biti djelomično odgovorna za različite funkcije dvaju proteina
Mechanisms of antitumour action of phytochemicals rutin and quercetin in colon cancer cells
No full textRak debelog crijeva (CRC) je jedan od vodećih uzroka smrti uzrokovanih
karcinomom u svijetu. Incidencija ovisi o geografskom položaju, načinu
života i genetskim faktorima. CRC se očituje stvaranjem adenomatoznih
polipa na unutarnjem zidu debelog crijeva. Liječenje CRC-a se odvija putem
operativnih zahvata, radijacije, kemoterapije ili kombinacije više opcija. 5-
FU je jedan od vodećih kemoterapeutskih opcija za liječenje CRC-a, ali zbog
česte pojave kemorezistencije tumorskih stanica, potrebno je razmatrati
nove opcije liječenja. Fitokemikalije pokazuju obećavajući potencijal
zahvaljujući svojem širokom spektru pozitivnih učinaka, među kojima je i
protutumorsko djelovanje. Cilj ovog rada je bio istražiti protutumorsko
djelovanje fitokemikalija rutina i kvercetina, te njihov učinak u kombinaciji
s 5-FU na tumorske stanice debelog crijeva HCT116. Korišteni su 24-satni
tretmani rutina, kvercetina, 5-FU i njihovih kombinacija, čije je djelovanje
ispitano korištenjem XTT-a, Western blota i imunofluorescencije. Rezultati
su pokazali citotoksičan učinak rutina i kvercetina koji je pojačan u
kombinaciji s 5-FU, aktivaciju autofagije te intrinzičnog i ekstrinzičnog puta
apoptoze, s pojačanim aktivacijama u kotretmanima s 5-FU, te utjecaj na
ERK/MAPK signalnu kaskadu i FoxO3a transkripcijski faktor. Pokazalo se da
su kombinacijske terapije učinkovitije od zasebnih, te da je rutin u
kombinaciji s 5-FU najučinkovitija kombinacija. Rutin, iako skromnijeg
djelovanja u zasebnom tretmanu, djeluje kao kemosenzitizator 5-FU-a,
pojačavajući njegov utjecaj na tumorske stanice. Ipak, potrebno je
provođenje istraživanja in vivo kako bi se pobliže ustanovilo djelovanje
fitokemikalija zasebno i u kombinaciji s kemoterapeuticima unutar
organizma.Colorectal cancer (CRC) is one of the leading causes of cancer-related death
worldwide. The incidence depends on geographical location, lifestyle, and
genetic factors. CRC is manifested through the formation of adenomatous
polyps on the inner wall of the colon. CRC is treated with surgery, radiation,
chemotherapy, or a combination of several options. 5-FU is one of the
leading chemotherapeutic options for the treatment of CRC, but due to the
frequent appearance of drug resistance in tumor cells, it is necessary to
consider new treatment options. Phytochemicals show promising potential
thanks to their wide spectrum of positive effects, among which is antitumor
activity. The aim of this work was to investigate the antitumor activity of
the phytochemicals rutin and quercetin, and their effect in combination with
5-FU in HCT116 colon tumor cells. 24-hour treatments of rutin, quercetin,
5-FU and their combinations were used, the effects of which were tested
using XTT, Western blot and immunofluorescence. The results showed the
cytotoxic effect of rutin and quercetin, which was enhanced in combination
with 5-FU, the activation of autophagy and the intrinsic and extrinsic
pathway of apoptosis, with increased activation in co-treatments with 5-FU,
and the impact on the ERK/MAPK signaling cascade and the FoxO3a
transcription factor. It has been shown that combination therapies are more
effective than separate therapies, and that rutin combined with 5-FU is the
most effective combination. Rutin, although of a more modest effect in a
separate treatment, acts as a chemosensitizer of 5-FU, enhancing its effect
on tumor cells. However, it is necessary to carry out research in vivo to
establish the action of phytochemicals separately and in combination with
chemotherapeutics inside the organism in a more detailed manner
Effect of olive leaf extract on glucose transporters in the brain of diabetic rats
No full textCiljevi istraživanja: Istražiti promjene u ekspresiji glukoznih transportera
GLUT1, GLUT2 i GLUT4 u mozgu zdravih, dijabetičkih i liječenih dijabetičkih
štakora polifenolima lišća masline.
Materijal i metode: U radu su korištene lijeve polutke mozga mužjaka Wistar
štakora podijeljenih u četiri skupine: C (Kontrola) – kontrolna skupina bez
tretmana; DMT1 – skupina s razvijenim dijabetesom nakon osam dana od
jednokratnog iniciranja streptozotocina (i.p., 60 mg/kg); DMT1+OLE –
skupina s razvijenim dijabetesom i posttretirana i.p. iniciranjem ekstrakta
polifenola lišća masline (OLE); DMT2 – skupina je hranjena visoko masnom
hranom tijekom 30 dana (standardna laboratorijska hrana obogaćena je
50 % palminim uljem). Skupine su sadržavale 3-5 životinja, starosti 2-3
mjeseca. Homogenati mozga korišteni su u WB analizi. Za procjenu
podataka korišten je program Statistica (softverski sustav za analizu
podataka), verzija 14 (TIBCO Software Inc., 2020., Palo Alto, CA, SAD).
Razlike između skupina procijenjene su jednosmjernom analizom varijance
(ANOVA One-way) praćenom post hoc Schefféovim testom. Regresijska
analiza (linearna regresijska analiza) korištena je za usporedbu međusobne
ovisnosti varijabli. Statistička značajnost određena je p vrijednosti manjom
od 0,05 (p<0.05) za faktor korelacije R.
Rezultati: U provedenim eksperimentalnim uvjetima, DMT1 i DMT2
pospješili su ekspresiju GLUT1, neznatno suprimirali GLUT2 i blago
eksprimirali GLUT4. Liječenje dijabetičnih životinja ekstraktom polifenola
lišća masline povećalo je ekspresiju GLUT1, nije utjecalo na ekspresiju
GLUT2 i GLUT4. U odnosu na DMT1, uočena je ekspresija GLUT1 u skupini
DMT2.
GLUT1 i GLUT2 statistički su se značajno mijenjali ovisno o istraživanim
skupinama (p=0,0055, odnosno p=0,0079). Statistički značajna razlika u
ekspresiji GLUT1 pronađena je između Kontrole i DMT1+OLE skupine
(p=0,0138) te između Kontrole i DMT2 skupine (p=0,0127). U odnosu na
Kontrolu, statistički značajna razlika u ekspresiji GLUT2 pronađena je u
DMT1 skupini (p=0,0174) i DMT2 skupini (p=0,0182). GLUT1 negativno je
korelirao s GLUT2 (r=-0,67) i β-aktinom (r=-0,68), GLUT2 sa GLUT4 (r=-
0,71), a pozitivno s β-aktinom (r=0,81). GLUT4 negativno je korelirao s βaktinom (r=-0,70). β-aktin statistički se značajno mijenjao ovisno o
skupinama (p=0,0000). U odnosu na Kontrolu, β-aktin je bio značajno
suprimiran u DMT1 skupini (p=0,0001), DMT1+OLE skupini (p=0,0002) i
DMT2 skupini (p=0,0000).
Zaključak: Na osnovu provedenog istraživanja utvrđena je važnost
određivanja glukoznih transportera mozga u dijabetesu i unosu
farmakoloških pripravaka. Visoke razine glukoze u DMT1 povećale su
ekspresiju GLUT1 koju terapija ekstraktom lišća masline, u tako kratkom
vremenu (sedam tretmana) nije smanjila.
Iako su naši rezultati suprotni onima u literaturi, smatramo da je za bolje
poznavanje regulacije glukoze putem glukoznih transportera potrebno
dodatno istraživanje koje omogućuju druge tehnike lokalizacije GLUT u
mozgu.Research Objectives: To study the changes in the expression of glucose
transporters GLUT1, GLUT2 and GLUT4 in the brain of healthy, diabetic and
diabetic rats treated with olive leaf polyphenols.
Material and Methods: The left cerebral hemispheres of male Wistar rats
were used for the work, which were divided into 4 groups: C (control) -
control group without treatment; DMT1 group with developed diabetes after
8 days of single administration of streptozotocin (i.p., 60 mg/kg); DMT1+
OLE - group with developed diabetes and post-treatment i.p. by
administration of olive leaf polyphenol extract (OLE); DMT2 group was fed
a high-fat diet for 30 days (standard laboratory diet enriched with 50%
palm oil). Groups consisted of 3-5 animals aged 2-3 months. Brain
homogenates were used for analysis of WB. Statistica (data analysis
software), version 14 (TIBCO Software Inc., 2020, Palo Alto, CA, USA) was
used for data analysis. Differences between groups were assessed using a
one-way analysis of variance (ANOVA one-way) followed by a post hoc
Scheffé test. Regression analysis (linear regression analysis) was used to
compare the interdependence of variables. Statistical significance was
determined with a p value of less than 0.05 (p < 0.05) for the correlation
factor R.
Results: Under the experimental conditions performed, DMT1 and DMT2
enhanced the expression of GLUT1, slightly suppressed GLUT2 and
expressed GLUT4 to a small extent. Treatment of diabetic animals with olive
leaf polyphenol extract increased the expression of GLUT1 but had no effect
on the expression of GLUT2 and GLUT4. Compared with DMT1, GLUT1
expression was observed in the DMT2 group.
GLUT1 and GLUT2 changed statistically significantly depending on the
groups studied (p=0.0055 and p=0.0079, respectively). A statistically
significant difference in GLUT1 expression was observed between the
control and DMT1+ OLE groups (p=0.0138) and between the control and
DMT2 groups (p=0.0127). Compared to control, a statistically significant
difference was found in GLUT2 expression in DMT1 group (p=0.0174) and
DMT2 group (p=0.0182). GLUT1 correlated negatively with GLUT2 (r=-
0.67) and β-actin (r=-0.68), GLUT2 with GLUT4 (r=-0.71), and positively
with β-actin (r=0.81). GLUT4 was negatively correlated with β-actin (r=-
0.70). β-Actin changed statistically significantly as a function of groups
(p=0.0000). Compared to control, β-actin was significantly suppressed in
DMT1 group (p=0.0001), DMT1+ OLE group (p=0.0002) and DMT2 group
(p=0.0000).
Conclusion: Based on the conducted research, the importance of
determining glucose transporters in the brain in diabetes and taking
pharmacological preparations was established. High glucose levels in DMT1
increased GLUT1 expression, which was not decreased by olive leaf extract
therapy in such a short time (7 treatments).
Although our results are in contrast to those in the literature, we believe
that further research should be conducted to better understand glucose
regulation by glucose transporters, allowing other GLUT localization
methods in the brain
Isolation and characterization of degradation products of lifitegrast obtained by forced degradation
No full textOvaj rad bavi se prisilnim studijama onečišćenja lifitegrasta, djelatne tvari
protuupalnog lijeka Xiidra koji se upotrebljava za liječenje sindroma suhog
oka. Lifitegrast je izlagan različitim ekstremnim uvjetima te je pri tome
praćena njegova stabilnost i razgradnja na pojedina onečišćenja. Provedene
su reakcije termolize, fotolize, hidrolize i oksidacije. Samim time, pomoću
kromatografskih i spektroskopskih tehnika, izolirani su i identificirani
produkti raspada lifitegrasta. U ovom će radu biti opisana kemijska svojstva
lifitegrasta, način provođenja stres testiranja te karakterizacija dobivenih
razgradnih produkata.Forced degradation studies of lifitegrast, the active ingredient in the antiinflammatory drug Xiidra, which is used to treat dry
eye syndrome are described. Lifitegrast was exposed to various extreme conditions in which its stability and degradation impurities were monitored. Thermolysis, photolysis, hydrolysis and oxidation reactions were carried out. By using chromatographic and spectroscopic techniques, the degradation products of lifitegrast were isolated, identified and characterized. In this work the chemical properties of lifitegrast, the methods of stress tests as well as the characterization of the obtained degradation products are discussed