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Alzheimer's disease: Pathophysiology and treatment
No full textAlzheimerova bolest je multifaktorijalna neurodegenerativna bolest koja
uzrokuje 60-80% svih demencija. Glavna karakteristika je nakupljanje
beta-amiloidnih plakova i neurofibrilarnih vretena u kori mozga, koje dovodi
do progresivnog opadanja kognitivnih funkcija. Alzheimerova se bolest
javlja u 2 oblika, sporadičnom i nasljednom, koji se razlikuju prema uzroku
te prosječnoj dobi početka bolesti. Zbog kompleksnosti, molekularni se
mehanizam nastanka objašnjava mnogobrojnim hipotezama od kojih su
najučestalije: hipoteza amiloidne kaskade, Tau hipoteza, kolinergička
hipoteza, hipoteza oksidativnog stresa, hipoteza upale te hipoteza
dishomeostaze metala. Upravo su na kolinergičkoj hipotezi temeljeni
lijekovi koji rade na principu inhibicije kolinesteraze, a trenutno su odobrena
3 takva lijeka: Donepezil, Galantamin i Rivastigmin. Navedeni lijekovi
spadaju u lijekove koji tretiraju simptome zajedno s Memantinom koji
funkcionira kao antagonist NMDA receptora. Druga skupina odobrenih
lijekova jesu lijekovi koji odgađaju kliničko opadanje, a trenutno je odobren
samo imunoterapeutik Aducanumab, koji djeluje uklanjanjem Aβ plakova.
Potaknuti navedenim istraživanjima, znanstvenici se sve više okreću drugim
hipotezama molekularnog mehanizma nastanka, uzimajući ih kao osnovnu
metu terapije pa se osim beta-amiloida istražuju i tau protein te upala i
oksidativni stres. Ovaj završni rad detaljno pojašnjava povezanost
navedenih hipoteza s patofiziologijom Alzheimerove bolesti te izlaže
postojeće terapijske opcije u svrhu boljeg razumijevanja njihovih
mehanizama djelovanja, čime se postavljaju osnove na kojima se temelje
daljnja istraživanja ove bolesti.Alzheimer's disease is a multifactorial neurodegenerative disorder which
causes 60-80% of all dementia. Main characteristic is progressive cognitive
decline caused by accumulation of β-amyloid plaques and neurofibrillary
tangles in cerebral cortex. There are 2 types of Alzheimer's disease, familial
and sporadic with differences in cause and age of onset. Due to disease
complexity, Molecular pathogenesis of Alzheimer's Disease is desribed by
several hypothesis, with main ones being: β-amyloid hypothesis, Tau
hypothesis, cholinergic hypothesis, hypothesis of oxidative stress,
inflammation hypothesis and metal dishomeostasis hypothesis. One of
these hypothesis, cholinergic hypothesis has set the base for drugs that
work as cholinesterase inhibitors. There are currently 3 approved
cholinesterase inhibitors used in treatment of Alzheimer's disease:
Donepezil, Galantamine and Rivastigmine. Those medications together with
NMDA receptor agonist Memantine are classified as drugs that treat
symptoms of Alzheimer's disease. Other type of treatment used are drugs
that delay clinical decline, with currently only immunotherapeutic
Aducanumab being approved for use. Sucess of this drug that binds and
removes Aβ plaques encouraged scientist to study other hypothesis and use
their findings as a targets in future therapies. New research, therefore use
not only β-amyloid as their main target but also tau protein, inflammation
and oxidative stress. This thesis gives detailed description of the connection
between hypothesis and pathofiziology of AD and presents existing
therapeutic options with purpose of better understanding their mechanism
of action, all in order to set the foundation on which future research will be
based
Characterization and effect of porphyrins on melanoma cells
No full textMelanom je zloćudni tumor kože koji nastaje prilikom maligne
transformacije melanocita, a zbog agresivnosti te metastatske prirode
predstavlja najsmrtonosniji oblik raka kože. Konvencionalne strategije u
liječenju raka poput radioterapije, kemoterapije i biološke terapije često
uzrokuju nuspojave te ne mogu nadvladati problem rezistencije tumora i
hipoksičnog tumorskog mikrookoliša. Jedno od inovativnih rješenja
predstavlja oblik minimalno invazivne i kliničke odobrene fototerapije,
fotodinamička terapija. Fotodinamička terapija temelji se na interakciji
svjetlosti, fotosenzibilizatora i kisika pri čemu dolazi do fotokemijske
reakcije i generiranja reaktivnih kisikovih vrsta koje zatim uzrokuju smrt
tumorskih stanica, oštećenje tumorskog krvožilja i potiču imunološki
odgovor.
Svrha ovog rada bila je ispitati djelotvornost piridilporfirina konjugiranih
lipofilnim lancima različitih duljina pri čemu su dva spoja N-oksidirana, a
dva N-metilirana. Mjerenjem intenziteta fluorescencije ispitana je količina
spojeva koji ulaze u stanice u različitim vremenskim intervalima,
fluorescentnom mikroskopijom praćena je lokalizacija porfirina u stanicama
te je pomoću MTT testa određena citotoksičnost porfirina u uvjetima
normoksije i hipoksije. Rezultati ispitivanja pokazali su kako se spojevi u
najvećoj količini nalaze u stanicama nakon 6 h inkubacije te da u većoj
količini ulaze u stanice fibroblasta (HDF). Također, pokazano je kako
porfirini u većoj količini ulaze u stanice pri 37 ℃ što bi moglo ukazati na
ulazak porfirina aktivnim transportom u stanice. Rezultati MTT testa
ukazuju na potencijalnu selektivnost porfirina za stanice melanoma (MEWO)
u koncentracijama 0.5 i 1 μΜ te pokazuju kako porfirini nisu citotoksični u
mraku. Potvrđeni su i rezultati prethodnih ispitivanja gdje je pokazano kako
veća lipofilnost spojeva utječe na bolji ulazak u stanicu te da N-oksidirani
spojevi potencijalno bolje djeluju u uvjetima hipoksije.Melanoma is a malignant skin tumor that arises during the malignant
transformation of melanocytes, and due to its aggressiveness and
metastatic nature, it is the deadliest form of skin cancer. Conventional
strategies in cancer treatment such as radiotherapy, chemotherapy, and
biological therapy often cause side effects and are not able to overcome the
problem of tumor resistance and hypoxia in the tumor microenvironment.
One of the solutions is photodynamic therapy (PDT), a form of minimally
invasive and clinically approved phototherapy. PDT is based on the
interaction of light, photosensitizer, and oxygen, whereby a photochemical
reaction occurs and the generation of reactive oxygen species is generated,
which then causes the death of tumor cells, damages tumor blood vessels,
and stimulates the immune response.
The aim of this work was to test the effectiveness of porphyrins conjugated
with lipophilic chains of different lengths, where two have N-oxidized and
two N-methylated pyridyl groups on meso position. The cell uptake of
porphyrins at different time points was determined by measuring the
fluorescence intensity, fluorescence microscopy was used to examine the
localization of porphyrins inside melanoma cell line, and the cytotoxicity of
porphyrins was determined under normoxia and hypoxia using the MTT
proliferation test. The results showed that the largest amount of the
porphyrins was found in the cells after 6 h of incubation, and in larger
amount in fibroblasts (HDF) in comparison to melanoma cell line (MeWO).
Also, it was shown that porphyrins enter the cells in larger quantities at
37 ℃, which could indicate the entry of porphyrins by active transport into
the cells. The results of the MTT test showed the potential selectivity of
porphyrins for melanoma cells (MEWO) and show that porphyrins do not
show dark toxicity in concentration up to 10 μM. The results of previous
tests were also confirmed, showing that the higher lipophilicity of the
compounds results in better cellular uptake and that N-oxidized compounds
potentially work better in hypoxi
The prevalence of Chlamydia trachomatis infection among women in Split and Dalmatia Country
No full textChlamydia trachomatis, sićušna obligatna unutarstanična gram-negativna
bakterija, najčešći je uzročnik bakterijskih spolno prenosivih bolesti u
svijetu. Kod 70% žena i 50% muškaraca infekcija ovom bakterijom javlja
se bez simptoma što uzrokuje odgođenu dijagnozu, neprestano širenje
zaraze te povećava rizik za razvoj dugoročnih problema s reproduktivnim
zdravljem. Najugroženija skupina je mlađa ženska seksualno aktivna
populacija (20-25 godina). Cilj provedenog istraživanja bio je odrediti
učestalost klamidijske infekcije kod žena Splitsko-dalmatinske županije te
prevalenciju infekcije kod rizične skupine (žene mlađe od 25 godina).
Također, na osnovu dobivenih rezultata zaključiti da li je potrebno uvođenje
metode probira kod najugroženije skupine. Uzorci 1000 asimptomatskih
žena analizirani su metodom realtime PCR.
Učestalost klamidijske infekcije u populaciji asimptomatskih žena Splitskodalmatinske županije iznosila je 2%. Pokazana je statistički značajna razlika
prevalencije infekcije kod žena mlađih od 25 godina, koje su rizična skupina
za komplikacije klamidijske infekcije, poteškoće u trudnoći i sterilitet, u
odnosu na žene starije od 25 godina. Također, pokazalo se kako drugi
mikroorganizmi u donjem dijelu spolnog sustava žena pogoduju
klamidijskoj infekciji, osobito statistički značajni podaci su bili infekcija HPVa s C. trachomatis. S obzirom da je klamidijska infekcija uglavnom
asimptomatska, a pravovremena dijagnoza i liječenje znatno smanjuju
prijenos zaraze i moguće komplikacije, preporuča se uvođenje metode
probira najugroženije populacije (mlađi od 25 godina). Infekcija
C.trachomatis je glavni preventibilni čimbenik razvoja steriliteta u žena
zbog čega je probir žena mlađih od 25 godina svrstan među najkorisnije i
najisplativije preventivne strategije kod steriliteta.Chlamydia trachomatis, a tiny obligate intracellular gram-negative
bacterium, is the most common cause of bacterial sexually transmitted
diseases in the world. In 70% of women and 50% of men, infection with
this bacterium occurs without symptoms which causes delayed diagnosis,
constant spread of infection and increases the risk of developing long-term
problems with reproductive health. The most vulnerable group is the
younger female sexually active population (20-25 years old). The aim of the
study was to determine the frequency of chlamydial infection among women
in Split and Dalmatia Country and prevalence of infection in the risk group
(women under 25 years of age). Also, on the basis of the obtained results,
conclude whether it is necessary to introduce a screening method for the
most vulnerable group. The samples of 1000 asymptomatic women were
analyzed by the realtime PCR method.
The prevalence of chlamydial infection in the population of asymptomatic
women in Split and Dalmatia Country was 2%. A statistically significant
difference in the prevalence of infection in women under 25 years of age,
who are a risk group for chlamydial infection complications, pregnancy
difficulties and sterility, was shown compared to women over 25 years of
age. Also, it was shown that other microorganisms in the lower part of the
female genital system favor chlamydial infection, particulary statistically
significent dana were HPV infection with C. trachomatis. Given that
chlamydial infection is mostly asymptomatic, and timely diagnosis and
treatment significantly reduce the transmission of infection and possible
complication, it is recommended to introduce a screening method for the
most vulnerable population (under 25 years of age). Chlamydial infection is
the main preventable factor in the development of sterility in women, which
is why the screening of women under 25 years of age is classified as one of
the most useful and cost-effective preventive strategies for sterility
Angiotenzin-konvertirajući enzim i njegov insercijsko/delecijski (I/D) polimorfizam u žena sa spontanim prijevremenim porodom
No full textThe angiotensin-converting enzyme (ACE) plays crucial role in regulating
blood flow to the placenta and promoting angiogenesis. Variations in the
ACE gene have been linked to adverse pregnancy outcomes, including
preterm birth (PTB), defined as delivery before 37 weeks of gestation. PTB
affects approximately 10.6% of live births worldwide, posing significant
health risks to newborns and long-term challenges for families. Despite its
prevalence, the specific mechanisms leading to PTB remain unclear. Results
of existing studies into the insertion/deletion (I/D) polymorphism of the ACE
gene have been inconsistent across different populations. To further
investigate this relationship, we conducted a case-control study on 120
Croatian women with PTB and 85 control subjects who delivered at term,
using polymerase chain reaction for genotyping. Additionally, we performed
comprehensive literature review conducted to gather studies that
investigated association of ACE I/D polymorphism and PTB, yielding a total
of six studies that we included in the meta-analysis. The case-control study
found no significant genetic differences between the PTB and control
groups. However, the meta-analysis showed a modest but statistically
significant association between the II and DD genotypes, suggesting that
individuals with the II genotype may have a lower risk of PTB. Although our
study suggests that the ACE I/D polymorphism does not significantly
contribute to PTB risk, the observed association in the meta-analysis
emphasizes the need for further research. Given the serious consequences
of PTB on neonatal health and family well-being, understanding genetic
factors like I/D ACE polymorphisms is crucial for developing targeted
interventions and improving maternal and neonatal outcomes.Angiotenzin-konvertirajući enzim (ACE) igra ključnu ulogu u regulaciji
protoka krvi prema posteljici i poticanju angiogeneze. Varijacije u ACE genu
povezane su s nepovoljnim ishodima trudnoće, uključujući prijevremeni
porod (PP), koji se definira kao porod prije 37. tjedna gestacije. PP zahvaća
približno 10,6% sve živorođene djece diljem svijeta, predstavljajući
značajan rizik za zdravlje novorođenčadi i dugoročne posljedice za obitelji.
Unatoč visokoj učestalosti PTB-a, točni mehanizmi koji do njega dovode još
uvijek nisu razjašnjeni. Dosadašnja istraživanja o povezanosti
insercijsko/delecijskog (I/D) polimorfizme ACE gena i PP-a dala su
nedosljedne rezultate u različitim populacijama. Kako bismo dodatno
istražili navedenu povezanost, proveli smo istraživanje na ukupno 120 žena
s PP-om i 85 kontrolnih ispitanica koje su rodile u terminu iz Hrvatske,
koristeći lančanu reakciju polimeraze za genotipizaciju. Dodatno, proveli
smo i sveobuhvatan pregled literature s ciljem pronalaženja svih do sad
provedenih istraživanja povezanosti ACE I/D polimorfizma i PP-a, koji je
rezultirao uključivanjem ukupno šest studija u meta-analizu. Istraživanjem
provedenim na Hrvatskoj populaciji nisu utvrđene značajne razlike u
frekvencijama genotipova i alela između skupine s PP-om i kontrolne
skupine. Međutim, meta-analiza je pokazala graničnu, ali statistički
značajnu povezanost pri usporedbi II i DD genotipova, sugerirajući da bi
osobe s II genotipom mogle imati manji rizik od PP-a. Iako naše istraživanje
pokazuje da ACE I/D polimorfizam ne doprinosi značajno riziku od PP-a,
uočena blaga povezanost u meta-analizi ukazuje na potrebu za daljnjim
istraživanjima. S obzirom na ozbiljne posljedice PP-a na zdravlje
novorođenčadi i dobrobit obitelji, razumijevanje genetičkih čimbenika,
poput I/D ACE polimorfizma ključno je za razvoj ciljanih intervencija i
poboljšanje ishoda za majku i dijete
Classification of disease status using machine learning on single cell RNA-sequencing data
No full textAlzheimerova bolest karakterizirana kao stanje progresivnog gubitka
kognitivnih funkcija, uključujući pamćenje, orijentaciju i prosuđivanje,
predstavlja vodeći oblik demencije u svijetu. Iako točan uzrok bolesti još
nije potpuno razjašnjen, ključni patološki mehanizmi bolesti uključuju
nakupljanje izvanstaničnih beta-amiloidnih plakova i hiperfosforiliranog Tau
proteina unutar živčanih stanica, što dovodi do neurofibrilarnih čvorova sa
posljedicom atrofije mozga i odumiranjem živčanih stanica te gubitkom
neurona i sinapsi. Cilj ovog diplomskog rada bio je istražiti potrencijalne
genetske markere i klasificirati status Alzheimerove bolesti pomoću
strojnog učenja, korištenjem podataka dobivenih RNA sekvenciranjem
pojedinačnih stanica (scRNA-seq). Visoko-propusno RNA sekvenciranje na
pojedinačnim stanicama revolucionarna je tehnika koja je omogućila
analizu cijelokupnog transkripcijskog profila pojedinačnih stanica u velikom
obujmu, što je ključna prednost u proučavanju heterogenosti stanica u
kompleksnim tkivima poput mozga. Za potrebe rada podatci RNA
sekvenciranja pojedinačnih stanica entorinalnog korteksa i superiornog
frontalnog girusa mozga post mortem (Braak stadiji 0, 2 i 6) bili su
prikupljeni i prethodno obrađeni korištenjem računalnog alata Seurat.
Uporabom alata strojnog učenja kao što su modeli logističke regresije i
nasumičnih šuma, analizirali smo podatke kako bi identificirali ključne gene
i signalne puteve povezane s progresijom bolesti. Analizom podataka
identificirali smo nekoliko gena kao potencijalne markere ovisno o stadiju
bolesti poput UTP8 gena za ranij stadij ili HNRNPH1, RPL10A i CLOCK gene
za kasniji stadij bolesti.Alzheimer’s disease, characterized by the progressive loss of cognitive
functions, including memory, orientation, and judgment, represents the
leading form of dementia worldwide.
Although the exact cause of the disease remains unclear, key pathological
mechanisms include the accumulation of extracellular beta-amyloid plaques
and hyperphosphorylated Tau protein within neurons, leading to
neurofibrillary tangles, brain atrophy, neuronal death, and the loss of
neurons and synapses. The aim of this thesis was to investigate potential
genetic markers and classify Alzheimer’s disease status using machine
learning, based on data obtained from single-cell RNA sequencing (scRNAseq). High-throughput single-cell RNA sequencing is a revolutionary
technique that has enabled the large-scale analysis of the entire
transcriptional profile of individual cells, which is a key advantage in
studying the heterogeneity of cells in complex tissues such as the brain.
For the purposes of this study, RNA sequencing data from single cells of
the entorhinal cortex and superior frontal gyrus of post-mortem brains
(Braak stages 0, 2, and 6) were collected and preprocessed using the
computational tool Seurat. Using machine learning tools such as logistic
regression models and random forests, we analyzed the data to identify
key genes and signaling pathways associated with disease progression. Our
analysis identified several genes as potential markers depending on the
disease stage, such as the UTP8 gene for earlier stages and the HNRNPH1,
RPL10A, and CLOCK genes for advanced stages
Fenotip rasta Saccharomyces cerevisiae taz1Δ: Utjecaj izvora ugljika i suplementacije masnim kiselinama
No full textBarth syndrome (BTHS) is an X-linked mitochondrial disorder caused by
mutations in the TAZ gene. This gene encodes tafazzin, an enzyme critical
for cardiolipin (CL) remodeling, essential for normal mitochondrial structure
and function. The absence of CL remodeling is the key pathological
mechanism responsible for BTHS. This research employs Saccharomyces
cerevisiae as a model organism to understand how different carbon sources
impact cellular processes, particularly focusing on non-fermentable sources
like pyruvate. The main objectives include characterizing growth defects in
tafazzin-deficient cells on various non-fermentable carbon sources and
determining the effect of fatty acid (FA) supplementation on cell growth.
Growth defects in taz1Δ and cld1Δtaz1Δ mutants on pyruvate indicate the
significance of specific carbon sources in cellular processes. FA
supplementation partially rescues these defects, suggesting potential
connections between FA metabolism, proper mitochondrial function, and
pyruvate utilization.Barthov sindrom (BTHS) je mitohondrijski poremećaj vezan uz Xkromosom uzrokovan mutacijom u TAZ genu. Ovaj gen kodira tafazzin,
enzim ključan za remodeliranje kardiolipina (CL), neophodnog za normalnu
strukturu i funkciju mitohondrija. Nedostatak remodeliranja CL ključni je
patološki mehanizam odgovoran za BTHS. U ovom se istraživanju koristi
Saccharomyces cerevisiae kao modelni organizam za razumijevanje kako
različiti izvori ugljika utječu na stanične procese, posebno se fokusirajući
na izvore ugljika poput piruvata. Glavni ciljevi istraživanja uključuju
karakterizaciju poremećaja rasta stanica kvasca s nedostatkom tafazzina
na različitim izvorima ugljika, te određivanje učinka suplementacije masnim
kiselinama (MK) na rast stanica. Poremećaji rasta u taz1Δ i cld1Δtaz1Δ
mutantima na hranjivim podlogama s piruvatom ukazuju na važnost
određenih izvora ugljika u staničnim procesima. Suplementacija MK
djelomično spašava ove poremećaje rasta, sugerirajući potencijalne veze
između metabolizma MK, pravilne funkcije mitohondrija i iskorištavanja
piruvata
Application of algae in technology and sustainable development
No full textNedostatak visokokvalitetnih prehrambenih proizvoda postaje globalni problem, a u budućnosti će se proizvodnja hrane trebati povećati za gotovo 90%. Alge su visokonutritivne te mogu pridonijeti rješenju ovog izazova, implementirajući se u funkcionalnu hranu. Alge, ključne za fotosintezu, podijeljene su na mikroalge i makroalge. Također, alge dijelimo prema njihovom klorofilu koji sadrže te koji im daje specifičnu boju, pa tako razlikujemo crvene, zelene i smeđe alge. Alge su prepoznate po svojim zdravstvenim prednostima, uključujući antioksidacijska, antibakterijska, antivirusna i neuroprotektivna svojstva. Različite vrste, poput Chlorelle vulgaris, nude poboljšanje glukoze u krvi te potporu mršavljenju. U industriji hrane za formiranje gelova i viskoznih otopina, možemo pronaći polisaharide algi kao što su agar, alginat i karagenan. U proizvodnji agara dominiraju alge Gelidium i Gracilaria te se u prehrambenoj industriji koristi oko 90% agara, a preostali dio se koristi u laboratorijima. Alginati i karagenani isto se koriste zbog svojstava gela te imaju široku primjenu u kozmetičkoj i farmaceutskoj industriji. Crvene alge poput Dulse i Nori nude esencijalne minerale i vitamine, dok smeđe alge kao Arame, Kombu, Morski špageti i Wakame pružaju velike količine makronutrijenata. S druge strane, zelene alge poput Chlorelle vulgaris, doprinose prehrambenoj industriji i istraživanju biogoriva. Drugae vrsta zelenih mikroalgi imaju različite pogodnosti, Dunaliella proizvodi β-karoten, a Haematococcus pluvialis astaksantin. Alge zbog svojih bioaktivnih spojeva imaju potencijalnu primjenu u farmaceutskoj industriji, posebno alginati koji se koriste u liječenju GERB-A, zacjeljivanju rana i dostavi lijekova. Nadalje, Chlamydomonas reinhardtii je istražena mikroalga s potencijalom u medicinskoj industriji i proizvodnji cjepiva, a rekombinantni proteini iz ove alge imaju perspektivnu primjenu u farmaceutskoj proizvodnji.The lack of high-quality food products is becoming a worldwide issue, and in the future, food production will need to increase by almost 90%. Algae, being highly nutritious, can contribute to addressing this challenge by being incorporated into functional food. Algae, crucial for photosynthesis, are classified into microalgae and macroalgae. They are further categorized based on their chlorophyll content, which gives them distinct colors, we distinguish red, green, and brown algae. Algae are renowned for their health benefits, including antioxidant, antibacterial, antiviral, and neuroprotective properties. Various types, such as Chlorella vulgaris, offer improvements in blood glucose and support weight loss. In the food industry, algae-derived polysaccharides like agar, alginate, and carrageenan are used for forming gels and viscous solutions. In agar production, Gelidium and Gracilaria algae dominate, with around 90% of agar used in the food industry, while the remain serves laboratories due to its thickening properties. Alginates and carrageenans find extensive use in the cosmetic and pharmaceutical industries due to their gel-forming characteristics. Red algae like Dulse and Nori provide essential minerals and vitamins, whereas brown algae such as Arame, Kombu, Sea Spaghetti, and Wakame offer substantial macronutrients. On the other hand, green algae like Chlorella vulgaris contribute to the food industry and biofuel research. Different species of green microalgae offer various benefits; for example, Dunaliella produces β-carotene, and Haematococcus pluvialis produces astaxanthin. Algae, rich in bioactive compounds, hold potential applications in the pharmaceutical industry, especially alginates used in treating GERD, wound healing, and drug delivery. Furthermore, Chlamydomonas reinhardtii, a researched microalga, shows potential in the medical industry and vaccine production. Recombinant proteins from this algae present promising applications in pharmaceutical manufacturing
Optimization of the method for quantification of glutathione in the model organism Drosophila melanogaster
No full textGlutation sudjeluje u održavanju fiziološke redoks ravnoteže kruženjem između reduciranog (GSH) i oksidiranog (GSSG) oblika. Omjer koncentracija reduciranog i oksidiranog oblika glutationa se koristi kao stanični i tkivni marker oksidativnog stresa.
Cilj ovog rada je bio optimizirati metodu za mjerenje glutationa. U radu su ispitani kemijski i biološki pristup mjerenja glutationa u ekstrahiranim uzorcima glava vinskih mušica. Kolorimetrijska metoda mjerenja glutationa koristi Ellman-ov reagens za determinaciju koncentracija reduciranog, oksidiranog i ukupnog glutationa u uzorku. Validnost metode je ispitana utjecajem kemijskih spojeva s prooksidansom, metamfetaminom (METH), i antioksidansom, kvercetinom (QUE). Biološkim pristupom mjerili smo pomak u fluorescenciji između reducirane i oksidirane forme roGFP-a kao odgovor na egzogeni reducens (DTT) i oksidans (DA).
Rezultati kolorimetrijske metode nisu pokazali statistički značajne razlike. Prisutan je trend povećanja GSH/GSSG u skupini tretiranoj s METH-om i njegovo smanjenje u skupini tretiranoj s QUE-om. Mjerenja biološkim pristupom GSH/GSSG nisu pokazala razlike između netretirane skupine i skupina tretiranih s oksidansom i reducensom.
Optimizacija metode za mjerenje reduciranog i oksidiranog oblika glutationa bila je djelomično uspješna. Obzirom na očekivane trendove dobivene kolorimetrijskom metodom smatramo da će daljnja optimizacija dovesti do pouzdanih i statistički značajnih rezultata. Korištenje roGFP indikatora nije optimalno za mjerenje redoks statusa u homogenatima tkiva već primarno za in situ mjerenja za koja je prvenstveno razvijena.Glutathione participates in maintaining the redox balance by cycling between the reduced (GSH) and oxidized (GSSG) forms. The ratio of concentrations of reduced and oxidized forms of glutathione is used as a cellular and tissue marker of oxidative stress.
The aim of this work was to optimize the method for quantification of glutathione. In the paper, the chemical and biological approach to measuring glutathione in extracted samples of the heads of fruit flies were examined. The colorimetric method of measuring glutathione uses Ellman's reagent to determine the concentrations of reduced, oxidized and total glutathione in a sample. The validity of the method was tested by the influence of chemical compounds with the prooxidant, methamphetamine (METH), and the antioxidant, quercetin (QUE). Using a biological approach, we measured the shift in fluorescence between the reduced and oxidized forms of roGFP in response to exogenous reductants (DTT) and oxidants (DA).
The results of the colorimetric method did not show statistically significant differences. There is a trend of increasing GSH/GSSG in the group treated with METH and its decrease in the group treated with QUE. Measurements using the biological approach of GSH/GSSG did not show differences between the untreated group and the groups treated with oxidant and reductant.
The optimization of the method for measuring the reduced and oxidized form of glutathione was partially successful. Considering the expected trends obtained by the colorimetric method, we believe that further optimization will lead to reliable and statistically significant results. The use of the roGFP indicator is not optimal for measuring redox status in tissue homogenates, but primarily for in situ measurements for which it was primarily developed
Razvoj i validacija LC-MS/MS metode za određivanje N-nitrozamina u farmaceutskim proizvodima
No full textN-nitrosamines are a class of organic compounds with a chemical structure that contains a nitroso (-NO) functional group attached to the amine nitrogen. Such an amine can have one or two alkyl groups attached to the nitrogen atom. According to various studies, most nitrosamines have been confirmed to be carcinogenic in various animal studies but potentially also in humans. For example, a higher incidence of gastric and oesophageal cancer has been described in people who were more exposed to nitrosamines in food. Additionally, another study reported that workers in the rubber industry who were exposed to increased levels of nitrosamines also had an increased incidence of mouth, oesophagus, and throat cancers. This mechanism is associated with cytochrome P450, which mediates the oxidation of nitrosamine compounds that then produce an alkylating agent that can react with genetic material.
Since June 2018, the US Food and Drug Administration (FDA), as well as the European Medicines Agency (EMA), have monitored the presence of nitrosamine contaminants in various drugs and introduced rules on maximum permissible levels. The aim of this work was to develop LC- MS/MS method(s) for the qualitative and quantitative determination of six nitrosamine contaminants in six different drugs on the market. The final forms of the drug (Finished Dosage From – FDF) and the standards of their active pharmaceutical ingredient (API) will be used during the development of the method. A review of the literature found several different methods regarding the technique of separation and analysis, as well as some methods that are used for several different pollutants. In this paper, the goal is to develop a method(s) adapted to as many potential nitrosamines as possible impurities in active substances.
N-nitrosamines, which are the subject of study in this work, must be well chromatographically separated from each other as well as from the main peak of the active substance being analysed. The N-nitrosamines of interest that will be studied are N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitroso-N-methyl-4-aminobutyric acid, N-nitrosoethylisopropylamine, N-nitroso-diisopropylamine and N-nitrosodibutylamine.
Medicines and standards of active substances used in this work are Azithromycin, Betahistine, Metformin, Metronidazole, Simvastatin, Sitagliptin and Vildagliptin in the form of various salts or free bases.
Two chromatographic methods were developed using three different chromatographic columns, and calibration curves were created for all combinations of methods and columns. Also, detection limits and quantification limits are determined for each nitrosamine contamination and for each method and type of column. For N-nitroso-N-methyl-4-aminobutyric acid, satisfactory detector sensitivity was achieved using only one method and one column.N-nitrozamini su klasa organskih spojeva s kemijskom strukturom koja sadrži nitrozo (-NO) funkcionalnu skupinu vezanu na dušik amina. Ovakav amin može imati jednu ili dvije alkilne skupine vezane na dušikov atom. Prema različitim provedenim studijama na životinjama, potvrđeno je da je većina nitrozamina kancerogena, a potencijalno je to slučaj i kod ljudi. Opisana je veća učestalost karcinoma želudca i jednjaka kod ljudi koji su više bili izloženi nitrozaminima u hrani. Osim toga, jedno drugo istraživanje navodi da radnici u gumarskoj industriji, koji su bili izloženi povećanim razinama nitrozamina, su također imali povećanu incidenciju karcinoma usne šupljine, jednjaka i grla. Ovaj mehanizam je povezan s citokromom P450 koji je posrednik oksidacije spoja nitrozamina koji potom proizvodi alkilirajuće sredstvo koje može reagirati s genetskim materijalom.
Od lipnja 2018. Američka agencija za hranu i lijekove (Food and Drug Administration, kao i Europska agencija za lijekove (European Medicines Agency) prate prisutnost nitrozaminskih onečišćenja u raznim lijekovima i uvode pravila o maksimalnim dopuštenim razinama. Cilj ovog rada bio je razviti kromatografsku, LC-MS/MS metodu(e) za kvalitativno i kvantitativno određivanje šest nitrozaminskih onečišćenja u šest različitih lijekova na tržištu. Kao materijal za analizu koristiti će se konačni oblici lijekova (gotove formulacije), a standardi njihovog aktivnog farmaceutskog sastojka (Active Pharmaceutical Ingredient - API) će se koristiti prilikom razvoja metode. Pregledom literature nađeno je više različitih metoda s obzirom na tehniku razdvajanja i analize, kao i neke metode koje se koriste za više različitih onečišćenja. U ovom radu cilj je razviti metodu(e) prilagođenu što većem broju potencijalnih nitrozaminskih onečišćenja u aktivnim supstancama.
N-nitrozamini, koji su predmet izučavanja u ovom radu, moraju se dobro kromatografski odvojiti međusobno, kao i od glavnog vrha aktivne supstance koja se analizira. N-nitrozamini od interesa koji će biti izučavani su N-nitrozodimetilamin, N-nitrozodietilamin, N-nitrozo-N-metil-4-aminomaslačna kiselina, N-nitrozoetilizopropilamin, N-nitrozo-diizopropilamin i N-nitrozodibutilamin.
Lijekovi i standardi aktivnih supstanci korištene u ovom radu su azitromicin, betahistin, metformin, metronidazol, simvastatin, sitagliptin i vildagliptin u obliku različitih soli ili slobodnih baza.
Razvijene su dvije kromatografske metode korištenjem tri različite kromatografske kolone, te su izrađene kalibracijske krivulje za sve kombinacije metoda i kolona. Također, za svako nitrozaminsko onečišćenje te za svaku metodu i vrstu kolone određene su granice detekcije i granice kvantifikacije. Za N-nitrozo-N-metil-4-aminomaslačnu kiselinu zadovoljavajuća osjetljivost detektora je postignuta korištenjem samo jedne metode i jedne kolone
The effect of pyridylporphyrins on mitochondria
No full textFotodinamička terapija (PDT) je novi terapeutski pristup za liječenje tumorskih bolesti te bakterijskih, gljivičnih i virusnih infekcija. PDT uključuje preferencijalni unos fotosenzibilizatora (PS) u ciljane stanice ili tkiva, nakon čega slijedi ozračivanje tog područja vidljivom svjetlošću. Nakon apsorpcije svjetlosti, PS prelazi u pobuĎeno stanje te generira reaktivne kisikove vrste (engl. reactive oxygen species, ROS). ROS reagiraju sa staničnim komponentama te uzrokuju staničnu smrt. Porfirini su prirodni intenzivno obojeni spojevi koji se zbog mogućnosti apsorpcije vidljive svjetlosti koriste kao fotosenzibilizatori. Protutumorski učinci fotodinamičke terapije uključuju tri glavna mehanizma uništavanja tumora, a to su oštećenje tumorskih krvnih ţila i sprječavanje daljnje angiogeneze, inicijacija lokalnog upalnog i imunosnog odgovora te direktna stanična smrt posredovana ROS-om. Od samih početaka eksperimentiranja s PDT-om, uočeno je da oštećuje mitohondrije, a primjećeno je i da kationski fotosenzibilizatori imaju povećan afinitet za kolokalizaciju s mitohondrijima. Iz tog razloga, u ovom radu cilj je bio ispitati učinak kationskih amfifilnih piridilporfirina na mitohondrije. Sintetizirani su 5-(4-acetamidofenil)-10,15,20-tris(1-oksidopiridin-3- il)porfirin, 5-(4-acetamidofenil)-10,15,20-tris(N-metilpiridin-3-il)porfirin triklorid, 5-(4-oktanamidofenil)-10,15,20-tris(1-oksopiridin-3-il)porfirin te 5-(4-oktanamidofenil)-10,15,20-tris(N-metilpiridin-3-il)porfirin triklorid. Struktura spojeva je potvrĎena NMR spektroskopijom, ali NMR spektri su pokazali prisutnost nečistoća koje se nisu uspjele ukloniti, stoga ovi spojevi nisu korišteni za daljnja biološka istraţivanja. Ispitan je učinak sličnog spoja, 5-(4-oktanamidofenil)-10,15,20-tris(N-metilpiridin-3- il)porfirin triklorida. Dokazano je da testirani spoj uspješno ulazi u stanice. Pokazana je i njegova toksičnost na mitohondrije analizom markera vanjske mitohondrijske membrane, ali ne i direktno nakupljanje porfirina u mitohondrijima. Nadalje, ispitano je da li navedena oštećenja dovode do stanične smrti te je dokazano da stanice koje su tretirane porfirinom, a VII potom osvijetljene, umiru apoptozom. Dakle, u ovom radu je dokazano da testirani spoj ulazi u stanice i uzrokuje oštećenje mitohondrija bez da direktno kolokalizira s njima te u konačnici dovodi do apoptoze stanica.Photodynamic therapy (PDT) is a new therapeutic approach for the treatment of tumor diseases, but also for bacterial, fungal and viral infections. PDT involves preferential uptake of a photosensitizer (PS), in the cells / tissues, followed by irradiation of the selected region by visible light. After the absorption of light, the PS reaches an excited state and generates reactive oxygen species (ROS). ROS react with cellular components and irreversibly modify them causing cell death. Porphyrins are natural, intensely colored compounds and are used as photosensitizers due to their ability to absorb visible light. The antitumor effects of photodynamic therapy include three major mechanisms of tumor destruction: damage to the tumor blood vessels and prevention of further angiogenesis, local inflammation and immune response, and direct cell death mediated by reactive oxygen species. From the beginning of experimentation with PDT, the damage to the mitochondria has been observed. Furthermore, it is known that cationic photosensitizers have an increased affinity for targeting the mitochondria. In this work, the aim was to investigate the effect of cationic amphiphilic tripyridylporphyrins on mitochondria. 5- (4-acetamidophenyl) -10,15,20-tris (1-oxidopyridin-3-yl) porphyrin, 5- (4-acetamidophenyl) -10,15,20-tris (N-methylpyridin-3-yl) porphyrin trichloride, 5- (4-octanamidophenyl) -10,15,20-tris (1- oxopyridin-3-yl) porphyrin and 5- (4-octanamidophenyl) -10,15,20-tris (N-methylpyridin-3) -il) porphyrin trichloride were synthesized. Their structure was confirmed by NMR spectroscopy, however, NMR spectra showed also impurities that could not be removed, thus these compounds were not used for further biological research. The effect of a similar compound, 5-(4-octanamidophenyl)-10,15,20-tris(N-methylpyridin-3- yl)porphyrin trichloride, was tested. The tested compound has shown successful internalization into cells. Its toxicity to the mitochondria has also been demonstrated by the analysis of markers of the outer mitochondrial membrane, but the direct accumulation of porphyrins in the IX mitochondria has not been shown. Ultimately, it was investigated if the damage that porphyrins are causing leads to cell death and it was proven that cells treated with porphyrin, after illumination, die by apoptosis. Thus, in this work, it is proven that the tested compound is able to enter cells, causes damage to the mitochondria, but does not colocalize with the mitochondria and ultimately leads to apoptosis