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Identifikacija, klasifikacija i kvantifikacija razvojnih stadija postnatalnog korteksa Monodelphis domestica pomoću metode izotropne frakcionacije
No full textOne of the less understood branches of modern neurobiology is the (in)ability of adult
mammals to regenerate their central nervous system (CNS) after injury. This is possible in
species such as octopuses and lizards but is lacking in mammals as it becomes lost during
development. Monodelphis domestica, the grey South American short‐tailed opossum is one
of the few mammals that can fully and functionally regenerate their CNS after injury in the
first two postnatal weeks, whereas most other species from the mammalian class lose their
regenerative capacity soon after birth. Opossum are born very immature and continue to
develop latched to the mother's belly, which therefore makes them excellent candidates for
neuroregenerative and neurodegenerative research.
Corticogenesis of the opossum brain is mostly postnatal, but in contrast to rodents has not
been sufficiently explored. To better understand the process of corticogenesis and transition
of heterogenous neural stem cells (NSC) into neurons, a specific method is required for
precise quantification of their number and characterization of their expression profile.
For this purpose, the isotropic fractionator (IFR) method has been modified and optimized for
work with opossum tissues and under inverted fluorescent microscope, and used to identify,
quantify, and classify cortical cell lines of the developing opossum brain. Data gained on fixed
and homogenized cortical tissue samples has been compared with the recently established
primary dissociated opossum cortical cell cultures, which also exhibit regenerative potential
during different developing periods in vitro. Additionally, the robustness and precision of the
IFR has been validated for opossum tissue by comparing the results with
immunohistochemically labelled cortical tissue slices and primary dissociated opossum
cortical cell cultures.
Three timepoints of interest have been chosen for research of postnatal development of the
opossum cortex. The timepoints have been previously examined on the opossum spinal cord,
which gave us the cerebral timepoints in conjunction with the knowledge that the maturation
process of developing spinal cords starts in cervical and ends in lumbar regions. The
timepoints chosen are: postnatal day 5‐6 (P5‐6), where the regeneration is still possible; P17‐18, where the regenerative properties cease, but the brain is still not fully developed; and
P30, where all the cortical structures are present, with a variety of cell lines.
We successfully developed IFR for opossum tissues at various postnatal ages and for the
inverted microscope, both as a one‐day and two‐day protocol variants. Additionally, we
showed that many of the existing antibodies can be efficiently used on opossum due to the
high protein homology between opossum and the immunogen/referent animal for which the
antibodies were developed.Jedna od najslabije istraženih grana moderne neurobiologije je (ne)mogućnost odraslih
sisavaca da regeneriraju središnji živčani sustav (SŽS) nakon ozljede. Regeneracija je moguća
u nižih vrsta kao što su hobotnice i gušteri, ali je rijetka u sisavaca, te se gubi tokom razvoja.
Monodelphis domestica, sivi južnoamerički kratkorepi oposum je jedan od rijetkih sisavaca
koji postnatalno mogu potpuno regenerirati SŽS nakon ozljede, zadržavajući svu njegovu
funkcionalnost. Takva regeneracija se može dogoditi unutar prva dva postnatalna tjedna, dok
većina ostalih sisavaca gubi mogućnost regeneracija odmah nakon poroda. Oposumi se rađaju
jako nerazvijeni i nastavljaju rast i razvoj pričvršćeni majci na trbuhu. Zbog tih osobina su
izvrsni kandidati za istraživanja neuroregeneracie i neurodegeneracije.
Kortikogeneza mozga oposuma odvija se većinom postnatalno, ali za razliku od glodavaca nije
dostatno istražena. Kako bi se bolje razumio proces kortikogeneze i tranzicija heterogenih
neuralnih matičnih stanica u neurone, potrebna je metoda s kojom se može dobiti precizna
kvantifikacija njihovog broja i karakterizacija njihove ekspresije proteina.
Metoda izotropne frakcionacije (IFR) je u tu svrhu modificirana, te je optimizirana za rad s
tkivima oposuma pod invertnim fluorescentnim mikroskopom. Koristi se za identifikaciju,
kvantifikaciju i klasifikaciju staničnih linija u korteksu oposuma u razvoju. Podatci dobiveni na
fiksiranim i homogeniziranim uzorcima tkiva korteksa su uspoređeni sa primarnim disociranim
staničnim kulturama korteksa oposuma, nedavno uspostavljenima u našem laboratoriju. One
također pokazuju regenerativne sposobnosti u različitim in vitro razvojnim periodima.
Robustnost i preciznost IFR metode je također validirana usporedbom rezultata sa
imunohistokemijskim prerezima tkiva korteksa i primarnim disociranim staničnim kulturama
korteksa oposuma.
Izabrali smo tri razvojna trenutka za istraživanje postnatalnog razvoja korteksa oposuma. Ti
razvojni trenutci su detektirani u istraživanjima leđne moždine oposuma in vivo i in vitro, s
posebnim naglaskon na to da maturacija SŽS počinje u cervikalnoj, a završava u lumbarnoj
regiji. Odabrani razvojni trenutci su postnatalni dan 5‐6 (P5‐6), kada je regeneracija SŽS još
uvijek moguća; P17‐18, kada regenerativne sposobnosti prestaju, ali mozak oposuma i dalje
nije razvijen do kraja; te P30, kada je prisutna definirana kortikalna struktura.
Uspješno smo razvili metodu IFR za tkiva oposuma različitih starosti za invertni mikroskop,
kao protokol za jedan i dva dana. Dodatno smo pokazali kako se mnoga postojeća antitijela
mogu koristiti na oposumima zbog visoke homologije između proteina u oposuma i
imunogene/referentne životinje za koju su antitijela napravljena
Proteinske promjene u leđnoj moždini neonatalnih oposuma tijekom razdoblja kada regeneracija prestaje biti moguća
No full textOne of the major challenges of modern neurobiology concerns the inability of the adult
mammalian central nervous system (CNS) to regenerate and repair itself after injury. It is poorly
understood why regenerative potential is lost with evolution and development and why it becomes
very limited in adult mammals. Even though our understanding of molecular and cellular
mechanisms that promote or inhibit neuronal regeneration is expanding, it is still unclear what are
the key differences between the neuronal systems that can and cannot regenerate, and how they
can be manipulated to change the outcome of the injury. Our incomplete understanding of
molecular events underlying neuronal development and regeneration is the main reason why
neurodegenerative diseases and brain and spinal cord injuries are still mostly incurable today.
A preferred model to study and reveal the cellular and molecular basis of regeneration is the
neonatal opossum (Monodelphis domestica). Opossums are marsupials that are born very
immature with the unique possibility to successfully regenerate spinal cord after injury in the first
two weeks of their life. After that, the regenerative capacity is abruptly lost: at 14 days in cervical
spinal segments and at 17 days in less mature lumbar spinal segments.
In this thesis, we used neonatal opossums to study molecular and cellular properties of spinal tissue
that has and does not have the capacity to regenerate after injury, to pinpoint the key differences.
Using a comparative proteomic approach, for the first time, we identified the proteins unique and
differentially distributed in the intact opossum spinal tissue with different regenerative capacities.
We have identified a total number of 4735 proteins involved in various cellular processes such as
cell growth, proliferation, differentiation, transcription, cell signaling, cytoskeleton and
extracellular matrix organization, axon guidance molecules, neurotrophic factors and entire
intracellular molecular pathways like mTOR and MAPK signaling pathway. We have also
identified many myelin associated proteins, known to act as inhibitors of CNS axon regeneration,
which was the positive control for our overall experimental procedure. Most interestingly, we have
identified a number of proteins related to different neurodegenerative diseases that change in the
opossum spinal cord during the period critical for neuroregeneration. The different distribution of
the selected proteins detected by comparative proteomics was further confirmed by Western blot,
and the changes in the expression of related genes were analyzed by quantitative reverse
transcription PCR. Furthermore, we explored the cellular localization of the selected proteins using
immunofluorescent microscopy. We showed that during the period of development when
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regeneration stops being possible, the substantial number of proteins known to be involved in
regeneration and development change their level in the opossum spinal cord. These results
upgraded the previous transcriptomic data about the genes differentially expressed in regenerating
and non-regenerating opossum spinal cord tissue. Comparison of the data obtained by genomic
and proteomic approaches allowed the identification of molecules of interest and narrowed the
number of candidates for functional assays.
For the first time, we successfully established primary neuronal spinal cell cultures from neonatal
opossums of different ages and different regenerative capacities, which represent a novel
mammalian in vitro platform, particularly useful to study CNS development and regeneration, and
to test the functional role of the candidate molecules, together with the intact neonatal opossum
spinal cord preparation. We have also developed the neuroregeneration scratch test to be
performed on primary neuronal spinal cell cultures deriving from P5 opossums.
Taken together, the results of this thesis contribute to a better understanding of neuronal
regeneration in mammals and identify candidate molecules as future targets to promote
neuroregeneration in mammalian CNS.Jedan od glavnih izazova suvremene neurobiologije je nemogućnost regeneracije središnjeg
živčanog sustava (SŽS) odraslih sisavaca nakon ozljede. Još uvijek nije jasno kako se i zašto
sposobnost regeneracije gubi tijekom evolucije i razvoja, te zašto regeneracija kod odraslih
sisavaca postaje iznimno ograničena. Iako se naše razumijevanje staničnih i molekularnih
mehanizama koji potiču ili inhibiraju regeneraciju zadnjih godina sve više produbljuje, još uvijek
je nejasno koje su to ključne razlike između živčanih sustava koji imaju i onih koji nemaju
sposobnost regeneracije te kako ih manipulirati sa ciljem da se promjeni ishod ozljede. Naše
nepotpuno razumijevanje molekularnih događaja koji su temelj razvoja i regeneracije živčanog
tkiva je glavni razlog zašto su neurodegenerativne bolesti, kao i ozljede mozga i leđne moždine
danas još uvijek neizlječive.
Prikladan model za proučavanje i otkrivanje stanične i molekularne osnove regeneracije su mladi
oposumi (Monodelphis domestica). Oposumi su tobolčari koji se rađaju nerazvijeni pa tijekom
prva dva tjedna svog života posjeduju jedinstvenu sposobnost potpune regeneracije leđne moždine
nakon ozljede. Nakon toga sposobnost regeneracije se naglo gubi: oko 14. dana u vratnom dijelu
leđne moždine, te 17. dana u njenom manje razvijenom lumbalnom dijelu.
Mlade oposume koristili smo za istraživanje molekularnih i staničnih svojstava tkiva leđne
moždine koje ima sposobnost regeneracije nakon ozljede, te onog koje nema, kako bi se odredile
ključne razlike. Uporabom komparativne proteomike, po prvi puta, identificirali smo proteine koji
su jedinstveni i različito izraženi u tkivu leđnih moždina oposuma koji mogu i onih koji ne mogu
regenerirati. Identificirali smo ukupno 4735 proteina koji sudjeluju u staničnom rastu, proliferaciji,
diferencijaciji, transkripciji, staničnoj signalizaciji, organizaciji citoskeleta i izvanstaničnog
matriksa, molekule koje usmjeravaju aksone, neurotrofne čimbenike i cijele unutarstanične
molekularne puteve kao što su mTOR i MAPK signalni put. Također, identificirali smo niz
proteina povezanih s mijelinom za koje se zna da djeluju kao inhibitori regeneracije aksona, što je
bila pozitivna kontrola za naš cjelokupni eksperimentalni postupak. Kao najvažniji rezultat, otkrili
smo da se mnoštvo proteina povezanih s neurodegenerativnim bolestima mijenja u leđnoj moždini
oposuma u vremenu kada regeneracija prestaje biti moguća. Različita distribucija odabranih
proteina dodatno je potvrđena Western blotom, a promjene u ekspresiji gena analizirane su
metodom kvantitativne reverzne transkripcije lančane reakcije polimeraze. Nadalje,
imunofluorescentnom mikroskopijom istražili smo staničnu lokalizaciju odabranih proteina.
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Pokazali smo da se tijekom razvoja, u trenutku kada regeneracija prestane biti moguća, znatno
mijenja razina proteina za koje se zna da kontroliraju regeneraciju i razvoj leđne moždine
oposuma. Dobiveni rezultati su nadogradili prethodna istraživanja vezana za gene različito
izražene u tkivu leđne moždine oposuma koje ima i onoga koje nema sposobnost regeneracije.
Također, usporedbom rezultata komparativne transkriptomike i proteomike moguće je bilo
identificirati molekule od interesa i suziti broj kandidata za funkcionalna istraživanja.
Po prvi puta, uspješno smo uspostavili primarne stanične kulture leđne moždine oposuma različite
dobi i različite regenerativne sposobnosti, koje predstavljaju novu in vitro platformu, posebno
korisnu za proučavanje razvoja i regeneracije SŽS-a, kao i za funkcionalna istraživanja molekula
kandidata u neuroregeneraciji, zajedno sa preparatom cjelovite leđne moždine oposuma
održavanog u kulturi. Također, uspostavili smo test neuroregeneracije koji uključuje mehaničku
ozljedu primarnih staničnih kultura dobivenih iz pet dana starih oposuma.
Zaključno, rezultati ovog istraživanja doprinose boljem razumijevanju regeneracije živčanog tkiva
sisavaca, te otkrivaju potencijalne terapijske molekularne mete koje bi mogle biti interesantne za
neuroregeneraciju SŽS-a sisavaca
Effect of Crocus sativus water extract on behavioral phenotypes and redox parameters of D.melanogaster induced
No full textOvisnost o metamfetaminu (METH) okarakterizirana je brojnim promjenama na razini neurona kao što je nastanak i nakupljanje reaktivnih kisikovih vrsta (ROS) i fluorescentnih završnih produkata naprednih glikacijskih reakcija (fAGEs). Drosophila melanogaster se već desetljećima koristi u istraživanju ovisnosti radi sličnosti sa sisavcima u bihevioralnom, monoaminiskom i molekularnom odgovoru na psihostimulanse.
U ovome radu istražen je utjecaj vodenog ekstrakta Crocus sativus (šafrana) koji je mužjacima administriran kroz hranu u razdoblju od sedam dana prije prisilnog ili svojevoljnog izlaganja METH-u u bihevioralnim testovima lokomotorne senzitizacije i preferencijalne konzumacije, nakon kojih su provedeni biokemijski eseji kvantificiranja koncentracije vodikovog peroksida (H2O2) i fAGEs-a u ekstraktima glave i tijela.
Potvrdili smo potencijal C.sativus da ublažava određene efekte koji nastaju kao posljedica konzumacije psihostimulansa. C.sativus djelovao je anksiolitički tako što je utjecao na smanjenje lokomotorne aktivnosti nakon drugog izlaganja D.melanogaster volatiliziranom metamfetaminu (vMETH). Mjerenjem koncentracije H2O2, nakon izlaganja vinskih mušica vMETH-u, primijećeno je antioksidativno djelovanje C.sativus čiji je predtretman spriječio njegovo nakupljanje u tijelima do kojega je došlo u kontrolnoj skupini vinskih mušica.
Mogućnosti vodenog ekstrakta C.sativus su brojne te se njegova svojstva trebaju dalje istražiti kako bi se u potpunosti iskoristio njegov potencijal. Govoreći o njegovom utjecaju na D.melanogaster induciranu metamfetaminom, naredna istraživanja trebaju se usmjeriti na različite starosne skupine vinskih mušica, ali i na prilagođavanje optimalne koncentracije ekstrakta C.sativus koja će imati najveći utjecaj na posljedice psihostimulansa.Addiction to methamphetamines (METH) is characterized by numerous changes at the level of neurons such as the generation and accumulation of reactive oxygen species (ROS) and fluorescent end products of advanced glycation reactions (AGEs). Drosophila melanogaster has been used for decades in addiction research due to its similarity with mammals in the behavioral, monoaminic and molecular response to psychostimulants.
In this paper, we investigated the influence of the aqueous extract of Crocus sativus (saffron), which was administered to males through food during the seven day period before forced or voluntary exposure to METH in behavioral tests of locomotor sensitization and preferential consumption. After that, biochemical assays were performed to quantify the concentration of hydrogen peroxide (H202) and fAGEs in head and body extracts.
We have confirmed the potential of C.sativus to alleviate certain effects that occur as a result of the consumption of psychostimulants. C.sativus had an anxiolytic effect by reducing locomotor activity after a second exposure of D.melanogaster to volatilized methamphetamine (vMETH). By measuring the concentrations of H202 after exposure of flies to vMETH, the antioxidant effect of C.sativus was observed, whose pretreatment prevented its accumulation in the bodies, which occurred in the control group of flies.
The possibilities of the water extract C.sativus are numerous and its properties need further research in order to fully exploit its potential. Speaking about its influence on D.melanogaster induced by methamphetamine, further research should focus on different age groups of flies, but also on adjusting the optimal concentrations of C.sativus extract that will have the greatest impact on the effects of psihostimulants
Platinum(IV) compounds as potential drugs: a quantitative structure-activity relationship study
No full textIntroduction: Machine learning methods, coupled with a tremendous increase in computer power in recent years, are promising tools in modern drug design and drug repurposing. Methods: Machine learning predictive models, publicly available at chemosophia.com, were used to predict the bioactivity of recently synthesized platinum(IV) complexes against different kinds of diseases and medical conditions. Two novel QSAR models based on the BiS algorithm are developed and validated, capable to predict activities against the SARS-CoV virus and its RNA dependent RNA polymerase. Results: The internal predictive power of the QSAR models was tested by 10-fold cross-validation, giving cross-R2 from 0.863 to 0.903. 38 different activities, ranging from antioxidant, antibacterial, and antiviral activities, to potential anti-inflammatory, anti-arrhythmic and anti-malarial activity were predicted for a series of eighteen platinum(IV) complexes. Conclusion: Complexes 1, 3 and 13 have high generalized optimality criteria and are predicted as potential SARS-CoV RNA dependent RNA polymerase inhibitors
EFFECT OF TREFOIL FACTOR 3 PROTEIN DEFICIENCY ON LIVER AND HIPPOCAMPUS OF MICE ON SHORT TERM HIGH FAT DIET TREATMENT
No full textTrefoil faktor protein 3 (Tff3) je mali peptid primarno eksprimiran u vrčastim stanicama probavila
gdje sudjeluje u zaštiti epitela od raznolikih štetnih utjecaja. Tff3 je prisutan i u jetri i u mozgu te
novija istraživanja ukazuju na potencijalno važnu ulogu u metaboličkim i neurodegenerativnim
poremećajima. Metaboličke bolesti kao što je Tip 2 dijabetes, i neurodegenerativne bolesti
uključujući Alzheimerovu bolest, dijele brojne patofiziološke poveznice. Proučavanje poveznica
u ranim fazama bolesti, prije uznapredovalih simptoma, bi moglo pridonijeti boljem razumijevanju
patogeneze ovih kompleksnih poremećaja i uspješnijem razvoju djelotvornih terapija. Stoga je cilj
ovog doktorskog rada bio istražiti ulogu Tff3 u jetri i hipokampusu u počecima aktivacije
patoloških procesa navedenih poremećaja uslijed kratkotrajnog tretmana visokomasnom hranom.
U tu svrhu je razvijen novi kongenični Tff3-/- mišji soj na C57BL/6N genskoj podlozi bez dodatnih
relevantnih metaboličkih mutacija. Zatim su novo-razvijeni Tff3-/- miševi oba spola te
odgovarajuće divlji tip kontrole hranjene visokomasnom hranom kroz 9 tjedana. Ispitan je
generalni metabolički status životinja, analizirana je ekspresija markera relevantnih
patofizioloških puteva u jetri te markera neurogeneze i neuroinflamacije u hipokampusu. Tff3-/-
životinje su imale statistički značajno manje mase i uočeno je smanjeno nakupljanje masti u jetri
u odnosu na WT kontrole što ukazuje na zaštitni učinak nedostatka Tff3. U jetri Tff3-/- mužjaka
je uočena značajno snižena razinu gena Il1α i Cxcr7, dok je u jetri Tff3-/- ženki povećana razina
Irs2, Atf4 gena te Ybx1 i Anp32a proteina. U hipokampusu Tff3-/- mužjaka snižena je razina
markera nezrelih neurona (Dcx) te markera mikroglija (Iba1), dok Tff3-/- ženke imaju povećanu
razinu Dcx te povećanu razinu markera zrelih neurona (NeuN). Analiza genske ekspresije markera
neurogeneze ukazuje na izraženije razlike u hipokampusu Tff3-/- ženki. U jetri i hipokampusu
životinja izloženih standardnoj hrani nisu detektirane statistički značajne razlike navedenih
promijenjenih markera što ukazuje kako se uočene promjene javljaju uslijed stresa izazvanog
visokomasnom hranom. Ovo istraživanje pruža novi uvid u uloge Tff3 proteina u jetri i
hipokampusu te pruža smjer za daljnja ispitivanja sa ciljem otkrivanja točnih mehanizama
djelovanja te potencijalno identifikacije novih terapijskih meta metaboličkih/neurodegenerativnih
poremećaja. Dodatno, ukazuje na potrebu za uključivanjem oba spola u buduća istraživanja s
obzirom da se uočen fenotip Tff3-/- mužjaka i Tff3-/- ženki bitno razlikuje.Trefoil factor protein 3 (Tff3) is a small peptide expressed mainly in goblet cells with a role
in healing and protection of epithelium. Recent research suggests that the Tff3 protein also
plays an important role in metabolic and neurodegenerative diseases. Metabolic diseases, such
as Type 2 diabetes and neurodegenerative diseases, such as Alzheimer's disease, share many
pathophysiological links. Studying these links in the early stages, before the onset of advanced
symptoms, could contribute to a better understanding of the pathogenesis of these complex
disorders and to the successful development of effective therapies. Therefore, the aim of this
doctoral thesis was to investigate the role of Tff3 in the liver and the hippocampus in the early
stages of pathological processes activation due to short-term treatment with a high-fat diet.
First, a new congenic Tff3-/- mouse strain was developed on the C57BL/6N genetic
background without additional relevant metabolic mutations. Subsequently, the newly
developed Tff3-/- mice of both sexes and corresponding wild-type controls were fed a highfat diet for 9 weeks. We examined the general metabolic status of the animals and analyzed
the expression of markers of relevant pathophysiological pathways in the liver and in the
hippocampus. Tff3-/- animals had significantly lower weight and reduced fat accumulation in
the liver compared with WT controls, suggesting a protective effect of Tff3 deficiency. The
results of molecular analysis showed decreased levels of the Il1α and Cxcr7 genes in the liver
of Tff3-/- males, whereas the levels of the Irs2 and Atf4 and Ybx1 and Anp32a were increased
in Tff3-/- females. In the hippocampus of Tff3-/- males, the protein level of the marker of
immature neurons (Dcx) and the marker of microglia (Iba1) were decreased, whereas levels
of Dcx and the marker of mature neurons (NeuN) were increased in Tff3-/- females. Analysis
of neurogenesis markers shows more pronounced effect in the hippocampus of Tff3-/- females.
No statistically significant differences in the aforementioned altered markers were detected in
both liver and hippocampus of the animals exposed to a standard diet, suggesting that the
observed changes are a consequence of stress induced by high-fat diet. This research provides
new insights into the role of Tff3 protein in the liver and the hippocampus and provides
direction for further investigations aimed at uncovering the precise molecular mechanisms. In
addition, it highlights the need to include both sexes in future research, as the observed
phenotype differs significantly by sex
Olive leaf extract affects oxidative stress in the brain of diabetic rats induced by streptozotocin
No full textCiljevi istraživanja: Istražiti promjene u ekspresiji antioksidacijskih enzima
SOD1, SOD2 i GPX1 u mozgu zdravih, dijabetičkih i dijabetičkih štakora
tretiranih polifenolima lišća masline.
Materijal i metode: U eksperimentalnom modelu SZT izazvanog dijabetesa
(dijabetes mellitus tip 1, DMT1) i HFD-izazvanog dijabetesa (dijabetes mellitus
tip 2, DMT2) korišten je veliki mozak mužjaka Wistar štakora podijeljenih u 4
skupine: C (Kontrola) - kontrolna skupina bez tretmana; DMT1-skupina s
razvijenim dijabetesom nakon 8 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 je obogaćena 50% s palminim uljem).
Skupine su sadržavale 3-5 životinja, starosti 2-3 mjeseca. 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 Scheffé-ovim testom i Kruskal-Wallis
test (višestruka usporedba p vrijednosti). Linearna regresijska analiza
korištena je za usporedbu međusobne ovisnosti varijabli u skupinama.
Statistička značajnost je određena sa p vrijednosti manjom od 0,05 (p<0.05)
za faktor korelacije R.
Rezultati: U provedenim eksperimentalnim uvjetima, Kruskal-Wallis test
pokazao je statistički značajnu razliku vrijednosti MDA između skupina DMT1
i DMT2 (p=0,0140).
Statistički značajna razlika ekspresije SOD2 pronađena je između skupina
DMT1 i DMT1+PF (p=0,011), što znači da terapija ima učinak na ekspresiju
mitohondrijskog SOD2. Analizom varijance pronađena je statistička
značajnost promjene ekspresije SOD2 svih ispitivanih skupina (p=0,009).
SOD2 je pokazao statistički značajnu pozitivnu korelaciju sa SOD1 (r=0,67) i
negativnu sa GPX1 (r=-0,74).
Zaključci: Istraživanjem ekspresije antioksidacijskih enzima SOD1, SOD2 i
GPX i razine MDA u mozgu dijabetičnih štakora nakon SZT izazvanog
dijabetesa (DMT1) i tretmana ekstraktom polifenola lišća masline utvrdili smo
razlike između ispitanih skupina. SOD2 je bio značajno eksprimiran u skupini
životinja liječenih ekstraktom polifenola lišća masline.
U skupini hranjenoj visoko-masnom hranom (DMT2) nismo pronašli značajne
razlike u odnosu na Kontrolu te razlike DMT1 i DMT2 u mozgu štakora.
Na uravnoteženje antioksidacijskog statusa mozga dijabetičnih životinja
vjerojatno ima utjecaj prisutni neenzimski antioksidansi topljivi u lipidima.Research objectives: To study the changes in the expression of antioxidant
enzymes SOD1, SOD2 and GPX1 in the brain of healthy, diabetic and diabetic
rats treated with olive leaf polyphenols.
Material and methods: In the experimental model of SZT-induced diabetes
(diabetes mellitus type 1, DMT1) and HFD-induced diabetes (diabetes mellitus
type 2, DMT2), the cerebrum of male Wistar rats was used, 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 (standard laboratory diet enriched with 50% palm oil) for
30 days. The groups consisted of 3-5 animals aged 2-3 months. 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 one-way analysis of variance (ANOVA one-way) followed by
post-hoc Scheffé test and Kruskal-Wallis test (multiple comparison of pvalues). Linear regression analysis was used to compare the interdependence
of variables in the groups. Statistical significance is determined with a p-value
of less than 0.05 (p < 0.05) for the correlation factor R.
Results: Under the experimental conditions conducted, the Kruskal-Wallis test
showed a statistically significant difference in MDA values between groups
DMT1 and DMT2 (p=0.0140).
A statistically significant difference in SOD2 expression was found between
the DMT1 and DMT1+ PF groups (p=0.011), indicating that the therapy has
an effect on mitochondrial SOD2 expression. Statistical significance of the
change in SOD2 expression in all groups studied was determined by analysis
of variance (p=0.009). SOD2 showed a statistically significant positive
correlation with SOD1 (r=0.67) and a negative correlation with GPX1 (r=-
0.74).
Conclusions: When the expression of the antioxidant enzymes SOD1, SOD2
and GPX and the MDA level in the brain of diabetic rats after SZT-induced
diabetes (DMT1) and treatment with olive leaf polyphenol extract were
examined, differences were found between the tested groups. SOD2 was
significantly expressed in the olive leaf polyphenol extract treated group.
In the group fed a high-fat diet (DMT2), we found no significant differences
compared to the control group and no differences between DMT1 and DMT2
in rat brain.
The balance of the antioxidant status of the brain of diabetic animals is
probably influenced by the presence of fat-soluble non-enzymatic
antioxidants
Uvođenje kvantitativnog PCRa za određivanja broja novosintetiziranih MCMV
No full textIn the research on virology, accurate and sensitive methods for detecting viral pathogens are essential. This study presents comparative analysis of two commonly used techniques, quantitative PCR (qPCR) and plaque assays, for the detection of murine cytomegalovirus (MCMV). Cytomegalovirus (CMV), a member of the Herpesviridae family, causes medical complications in immunocompromised individuals including congenital defects, post-transplant graft rejections and cardiovascular diseases. CMV infection is complex due to its ability to establish latency in its host and capacity to establish lifelong infections. Understanding its dynamics, especially in immunocompromised individuals, is important. The paper presented shows the efficacy of qPCR as a tool for MCMV quantification, presenting its capability to detect viral DNA even in latent or non-active states. Our findings show a considerable growth in sensitivity when using qPCR compared to the conventional plaque assay. qPCR yielded higher viral particles concentrations and demonstrated advantages in terms of precision and quantification accuracy. qPCR sensitivity allows the detection of minimal quantities of MCMV genome, giving us a better understanding of the virus's latent phase and improving our ability to measure viral dynamics. Furthermore, qPCR's ability to provide results with a faster processing time and reduced subjectivity compared to plaque assays presents it as a useful tool for research and clinical applications. The study underlines the potential of qPCR to improve MCMV detection and quantification, showing a better approach in studying the virus, evaluating antiviral treatments, and eventually improving our ability to treat CMV-related diseases.U istraživanjima u području virologije, precizne i osjetljive metode za otkrivanje virusnih patogena su esencijalne. Ova studija predstavlja komparativnu analizu dviju često korištenih tehnika, kvantitativnog PCR (qPCR) i testova plakova, za detekciju mišjeg citomegalovirusa (MCMV). Citomegalovirus (CMV), član obitelji Herpesviridae, uzrokuje medicinske komplikacije kod imunokompromitiranih pojedinaca uključujući urođene defekte, odbacivanje presatka nakon transplantacije i kardiovaskularne bolesti. CMV infekcija složena je zbog svoje sposobnosti uspostavljanja latencije u svom domaćinu i sposobnosti uspostavljanja cjeloživotnih infekcija. Razumijevanje njegove dinamike, posebno u imunokompromitiranih osoba, iznimno je važno. Predstavljeni rad pokazuje učinkovitost qPCR-a kao metode za kvantifikaciju MCMV-a, prikazujući njegovu sposobnost detekcije virusne DNA čak i u latentnom ili neaktivnom stanju. Naši rezultati pokazuju značajan porast osjetljivosti pri korištenju qPCR-a u usporedbi s konvencionalnim testovima plakova. qPCR detektirao je veće koncentracije virusnih čestica i pokazao prednosti u pogledu preciznosti i točnosti kvantifikacije. Osjetljivost qPCR-a omogućuje otkrivanje minimalnih količina MCMV genoma, što nam daje bolje razumijevanje latentne faze virusa i poboljšava našu sposobnost mjerenja dinamike virusne infekcije. Nadalje, sposobnost qPCR-a da pruži rezultate s bržim vremenom obrade i smanjenom subjektivnošću u usporedbi s analizama plakova predstavlja ga kao koristan alat za istraživanja i kliničke primjene. Studija naglašava potencijal qPCR-a za poboljšanje detekcije i kvantifikacije MCMV-a, predstavljajući bolji pristup u proučavanju virusa, procjeni antivirusnih tretmana i na kraju poboljšanju naše sposobnosti liječenja bolesti povezanih s CMV-om
Suplementacija amino kiselinama djelomično spašava poremećaj rasta stanica kvasaca s nedostatkom Taz1
No full textBarth syndrome is a rare recessive genetic disorder. It mostly affects
mitochondria-rich organs like the heart and skeletal muscles. However,
there is a wide range of symptoms like cardiomyopathy, skeletal
myopathy, hypertrophy, fibrosis, increased urinary excretion of 3-
methylglutaconic acid, learning disabilities, attention deficit and so on. The
disorder is caused by a mutation in the tafazzin (TAZ) gene on the X
chromosome. TAZ gene codes for the tafazzin enzyme that participates in
a mechanism of cardiolipin (CL) maturation. CL is a phospholipid located
in the inner mitochondrial membrane. It provides membrane homeostasis
and cell integrity. CL mutations make mitochondrial energy production less
effective and shorter cell life. Since the physiology behind CL synthesis is
not completely understood patients with Barth syndrome are treated
symptomatically. However, with a better understanding of biochemistry
and molecular mechanism connected to CL, new treatment methods could
be considered. One of them is a high-protein diet. Proteins are
macromolecules comprised of long chains of amino acids. Amino acids are
building blocks for a variety of compounds. In some research amino acid
supplementation improves cell growth. That is why we are interested in
what effects supplementation of amino acids has on TAZ-deficient S.
cerevisiae cells.Barthov sindrom je rijetka recesivna genetska bolest. Najčešće zahvaća
organe bogatije mitohondrijima poput srca i skeletnih mišića. Ipak, bolest
karakteriziraju različiti simptomi poput: kardiomiopatije, skeletne
miopatije, hipertrofije, fibroze, povećanog mokrenja 3-metilglutakonske
kiseline, poteškoća u učenju, nedostatk pažnje i tako dalje. Poremećaj
nastaje mutacijom tafazzin (TAZ) gena na X kromosomu. TAZ gen
generira tafazzin enzim koji sudjeluje u mehanizmu sazrijevanja
kardiolipina (CL). CL je fosfolipid koji se nalazi u unutarnjoj membrani
mitohondrija. Održava homeostazu membrane i stanični integritet.
Mutacije CL-a smanjuju mitohondrijsku proizvodnju energije i životni vijek
stanica. Kako fiziologija sinteze CL-a nije upotpunosti jasna Barthov
sindrom se liječi simptomatski. Međutim, s boljim razumijevanjem
biokemije i molekularnih mehanizama povezanih s CL-om, mogu se
razmatrati nove metode liječenja. Kao što je visokoproteinska prehrana.
Proteini su makromolekule građene od dugih aminokiselinskih lanaca.
Amino kiseline su gradivne jedinice mnogih tvari. U nekim istraživanjima
suplementacija amino kiselinama uzrokuje stanični rast. Zbog toga smo
zainteresirani kako suplementacija amino kiselina utjeće na S. cerevisiae
stanica bez TAZ gena
Uloga proteina ADAR1 u replikaciji humanoga herpes virusa 8 (HHV-8)
No full textJedna od najčešćih posttranskripcijskih modifikacija na dvolančanoj RNA (dsRNA) kod ljudi jest proces prelaska adenozina u inozin (A-I uređivanje). Deaminaciju kataliziraju adenozin deaminaze koje djeluju na RNA (ADAR). ADAR geni kodiraju četiri izoforme ADAR proteina: ADAR1- p110, ADAR1-p150, ADAR2 i ADAR3. Istraživanja su pokazala da je ekspresija ADAR1 na uređenim mjestima najviša zbog čega se smatra da ima ključnu ulogu u promjenama u ekspresiji RNA kao i njezinoj funkciji. Uređivanje A-I putem ADAR-a ima velik učinak na očuvanje stanične homeostaze, dok se abnormalnosti u uređivanju povezuju s patogenezom brojnih bolesti, uključujući autoimune bolesti i tumore. ADAR svoju ključnu ulogu pokazuje u regulaciji imunološkoga odgovora domaćina i kontroliranju replikacije virusa. Ovisno o specifičnome virusu te kombinaciji stanica domaćina, uređivanje može imati provirusne ili antivirusne učinke. Kaposijev sarkom povezan s herpesvirusom (KSHV) primjer je virusa kod kojega ADAR1 ima provirusni učinak. KSHV je dvolančani DNA virus povezan s Kaposijevim sarkomom (KS), primarnim efuzijskim limfomom (PEL) i multicentričnim Castlemanovom bolešću (MCD) koji tijekom svojega životnog ciklusa prolazi kroz dvije faze: latentnu i litičku. Tijekom latentne faze ekspresija gena je uvelike ograničena. Uređivanje jednoga od transkripata ekspresioniranoga u latentnoj fazi, K12 RNA, s pomoću ADAR1 eliminira njegov onkogeni potencijal. Nakon reaktivacije KSHV-a slijedi litička faza kada se ekspresija gena povećava i aktivira se urođeni imunološki odgovori domaćina koji sprječava replikaciju virusa. S obzirom na to da je reaktivacija povezana s aktivnošću interferona (IFN) te ADAR1 utječe na nju, dovodi se u pitanje ako ADAR1 kontrolira aktivaciju signalnoga puta receptora sličnih RIG-u (RLR). Potvrđeno je da povećane razine ADAR1 proteina smanjuje urođeni imunološki odgovori na RNA što utječe na proliferaciju virusa.One of the most common post-transcriptional modifications of double-stranded RNA (dsRNA) in humans is the conversion of adenosine to inosine (A-I editing). Deamination is catalyzed by RNA-acting adenosine deaminase (ADAR). ADAR genes encode four isoforms of ADAR proteins. ADAR1-p110, ADAR1-p150, ADAR2, and ADAR3. According to studies, ADAR1 expression is more abundant in regulatory locations, indicating that ADAR1 is crucial for modifying RNA expression and function. The cellular homeostasis-maintaining role of A-I editing by ADARs is significant, while abnormal editing is thought to play a role in the development of a wide range of illnesses, including cancers and autoimmune diseases. ADARs are crucial for controlling viral replication and modulating host immunological responses. Editing can have proviral or antiviral effects, depending on the particular virus and host cell combination. A virus in which ADAR1 has a proviral effect is Kaposi's sarcoma-associated herpesvirus (KSHV). KSHV is a double-stranded DNA virus that goes through two stages of its life cycle: latent and lytic phase. It is linked to Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD). During latency, gene expression is severely restricted. Editing of K12 RNA, one of the latently expressed transcripts, by ADAR1, eliminates its oncogenic potential. KSHV reactivation is followed by a lytic phase, during which the host's innate immune response is triggered and gene expression rises, allowing the virus to proliferate. Since reactivation of the RIG-like receptor (RLR) signaling pathway is linked to IFN activity, which ADAR1 affects, it has been questioned if ADAR1 regulates it. It has been shown that elevated ADAR1 protein levels diminish innate immune responses to RNAs that affect viral replication
