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Olfaktorne promjene kvantificirane Testom za identifikaciju mirisa Sveučilišta u Pennsylvaniji: Preliminarno istraživanje o utjecaju traumatske ozljede kralježnične moždine na olfaktornu funkciju u ljudi.
No full textTraumatic spinal cord injury (SCI) is a disruptive neurological condition which severely affects the lives of individuals. SCI leads to partial or complete impairment of movement below the level of the lesion and is accompanied by a cascade of events resulting in the disruption of many neurological pathways. Among other complications, one study reported that people living with SCI can experience olfactory impairment. Although smell is a sense people consider non-essential, its importance is highlighted after experiencing difficulties with olfaction. Olfactory impairment can lead to feelings of vulnerability, emotional and mental problems, and an inability to detect real dangers such as spoiled food or gas leaks. This study aimed to test olfactory function in individuals who suffered traumatic SCI using the University of Pennsylvania Smell Identification Test (UPSIT). UPSIT is a standardized 40-question multiple-choice test that utilizes the odour microencapsulation technology and is widely used for assessing chemosensory dysfunction. Since it is culture-specific, the Italian version was chosen for administration to the Croatian population since it did not contain unfamiliar odours found in other versions. 20 participants with traumatic SCI were tested regardless of their gender, age, levels of the lesion and extent of the injury (complete or incomplete). The obtained scores were compared to the diagnosis scale which comes with the test. 19 out of 20 participants (95%) had reduced olfactory function and most of them fell under the category “Mild Microsmia”. One participant claimed to have lost both a sense of smell and a sense of taste after the injury. Interestingly, when asked to assess their own olfactory function, participants evaluated it as very good or excellent. In conclusion, this study found that a large proportion of individuals living with SCI have an olfactory impairment and that the majority of them are not aware of it.Traumatska ozljeda leđne moždine (SCI) je disruptivno neurološko stanje koje značajno utječe na kvalitetu života pojedinaca. Ozljede leđne moždine dovode do djelomičnog ili potpunog oštećenja ispod razine lezije i popraćene su kaskadom događaja koji dovode do poremećaja mnogih neuroloških puteva. Među ostalim problemima, u jednoj studiji je prethodno zabilježena korelacija između olfaktornog oštećenja i SCI-ja. Iako je miris osjet kojeg većina smatra najmanje bitnim, njegova važnost se posebno ističe nakon njegova gubitka. Gubitak osjeta mirisa može dovesti do osjećaja ranjivosti, emocionalnih i psihičkih problema, kao i do nemogućnosti prepoznavanja stvarnih opasnosti poput pokvarene hrane ili curenja plina. Cilj ovog rada bio je testirati olfaktornu funkciju u pojedinaca koji su pretrpjeli traumatsku ozljedu leđne moždine pomoću Testa za identifikaciju mirisa Sveučilišta u Pennsylvaniji (UPSIT). UPSIT je široko primjenjivani standardizirani test od 40 pitanja s 4 ponuđena odgovora razvijen za procjenu kemosenzorne disfunkcije i temelji se na tehnologiji mikroenkapsulacije. Budući da postoje kulturno-specifične verzije istog testa, za testiranje je odabrana talijanska verzija jer nije sadržavala mirise nepoznate hrvatskoj populaciji. 20 ispitanika s traumatskom ozljedom leđne moždine testirano je neovisno o njihovom spolu, dobi, razini lezije i kompletnosti ozljede (potpuna ili nepotpuna). Dobiveni rezultati uspoređeni su s ljestvicom dijagnoze olfaktorne funkcije koja dolazi uz sam test. U 19 od 20 ispitanika (95%) uočena je smanjena olfaktorna funkcija, pri čemu je najviše ispitanika pripalo u kategoriju „blage mikrosmije”. Jedan ispitanik je tvrdio da je nakon ozljede potpuno izgubio i osjet mirisa i osjet okusa. Zanimljivo, kada ih se pitalo kakvom bi oni procijenili svoju olfaktornu funkciju, većina ispitanika ocijenila je svoj osjet mirisa vrlo dobrim ili odličnim. U zaključku, ovim istraživanjem pokazalo se da većina ljudi koja živi s ozljedom kralježnične moždine ima smanjenu olfaktronu funkciju i da nje često nisu svjesni
Synthesis of BODIPY compounds that target intracellular organelles
No full textFluorescentna mikroskopija je moćna tehnika slikovnog prikazivanja
koja se koristi u različitim znanstvenim područjima, posebno u biologiji i
medicini, kako bi se vizualiziralo i proučavalo raspodjelu i ponašanje molekula
unutar stanica i tkiva. Koristi se fenomenom fluorescencije, koji omogućuju
različiti fluorofori ili fluorescentne boje. Kao dio ovog rada, sintetizirane su
dvije nove fluorescentne BODIPY boje koje sadrže supstituente na mesopoložaju, koji su posebno dizajnirani da ciljaju lizosome ili endoplazmatski
retikulum (ER). Sama sintetetska strategija temeljila se na pripremi BODIPY
karboksilne kiseline, koja je metodom peptidne sinteze u izvrsnim prinosima
vezana za derivate morfolina ili tosilsulfonamida. Pokušaji sinteze BODIPY
derivata s trifenilfosfonijevom soli koja bi ciljala mitohondrije nisu uspjeli zbog
neuspješne Williamsove sinteze etera BODIPY fenola s bromoalkinom.
Spektralna i fotofizička svojstva istražena su u nepolarnim (CH2Cl2), polarnim
otapalima - aprotičnim (CH3CN) i protičnim (H2O). Spojevi imaju usku i
snažnu apsorpcijsku (≈ 498 nm) i emisijsku vrpcu (510-515 nm) u vidljivom
području te male Stokesove pomake. Spojevi ne pokazuju fluorosolvatokromna svojstva, ali kvantni prinosi fluorescencije (Φf = 0,50-0,75)
opadaju s povećanjem polarnosti otapala. Vremena života singletnih
pobuđenih stanja izmjerena su metodom vremenski koreliranog brojanja
pojedinačnih fotona i nalaze se u rasponu nekoliko nanosekundi (τ = 2,6-3,0
ns). Eksperimenti fluorescentne mikroskopije izvedeni su na staničnoj liniji
HFF, a pokazuju laku internalizaciju spojeva i specifično bojenje lizosoma ili
ER-a pri koncentracijama od 5 μM, odnosno 1 μM. Dobiveni rezultati sugeriraju
moguću primjenu novosintetiziranih BODIPY spojeva u medicini i biologiji kao
fluorescentnih indikatora za ciljanje lizosoma i ER-a.Fluorescence microscopy is a powerful imaging technique used in
various scientific fields, particularly in biology and medicine, to visualize and
study the distribution and behavior of molecules within cells and tissues. It
exploits the phenomenon of fluorescence, which is attributed to various
fluorophores or fluorescent dyes. As a part of this thesis, two new fluorescent
BODIPY dyes were synthesized, containing substituents at the meso-position,
specifically designed to target lysosomes or endoplasmic reticulum (ER). The
synthetic strategy was based on the preparation of a BODIPY carboxylic acid,
which was in the peptide coupling protocol in excellent yields attached to a
morpholine residue or tosylsulphonamide derivative, respectively. Attempted
synthesis of a BODIPY derivative with triphenylphosphonium salt to target
mitochondria failed due to unsuccessful Williams ether synthesis of a BODIPY
phenol with a bromoalkyne derivative. Spectral and photophysical properties
were investigated in nonpolar (CH2Cl2), polar solvents – aprotic (CH3CN) and
protic (H2O). The compounds have narrow and strong absorption (≈ 498 nm)
and emission bands (510-515 nm) in the visible region and have small Stokes
shifts. The compounds do not exhibit fluoro-solvatochromic properties, but the
quantum yields of fluorescence (Φf = 0.50-0.75) decrease with increasing
solvent polarity. Singlet excited state lifetimes were measured by timecorrelated single photon counting and they are in the nanosecond time-scale
(τ = 2.6-3.0 ns). The fluorescence microscopy experiments were performed
on the HFF cell line, demonstrating the easy internalization of the compounds
and specific staining of lysosomes or ER at concentrations 5 μM and 1 μM,
respectively. The obtained results suggest a potential application of the newly
synthesized BODIPY compounds in medicine and biology as fluorescent
indicators for targeting lysosomes and ER
Dopamin I njegova interakcija sa DISC1 u životinjskim modelima
No full textDISC1 is a scaffold protein described as a risk factor for major mental illnesses, including schizophrenia. It was discovered in Scottish family 23 years ago. The gene was identified at the breakpoint on chromosome 1 of the balanced translocation (1;11) (q42.1; q14.3). In the last few decades there has also been an accent on the role of dopamine in pathophysiology of mental illnesses, specifically the dopamine receptors. Since then, different hypotheses have surfaced about its involvement in those illnesses. One of the main theories is the ‘Dopamine hypothesis of schizophrenia’. It described an impact of DISC1 in regulation of dopamine receptors in the striatum. DISC1 affects the receptors regulation, GSK3 pathway for D2R and KLF16 gene for D2R, and causes excessive transmission of dopamine. Increased dopamine synthesis has been related to negative symptoms of schizophrenia. For studying the pathology of DISC1 related illnesses scientists use rodent models. Their genetic, biological and behaviour characteristics closely resemble those of humans which makes them great candidates for drug development.DISC1 je _ protein opisan kao jedan od glavnih faktora rizika u razvoju mentalnih bolesti, uključujući shizofreniju. Otkriven je prije 23 godine u jednoj škotskoj obitelji. Gen je identificiran na kromosomu 1 uravnotežene translokacije (1;11) (q42.1; q14.3). U zadnja dva desetljeća je također bio naglasak na ulozi dopamina u patozifiologiji mentalnih bolesti, točnije dopaminskih receptora. Od tada su se pojavile različite hipoteze o njegovom značaju u mentalnim bolestima. Jedna od glavnih teorija je ‘Dopaminska hipoteza shizofrenije’. Ona opisuje utjecaj DISC1 proteina na regulaciju dopaminskih receptora u striatumu. DISC1 utječe na regulaciju receptora, GSK3 signalni put za D2R I KLF gen za D1R, te uzrokuje pretjeranu transmisiju dopamine. Povećano lučenje dopamine se povezuje sa negativnim simpromima shizofrenije. Za proučavanje patologije DISC1 povezanih bolesti koriste se modeli glodavca. Njihove genetske i biološke karakteristike, te karakteristike ponašanja su slične kao kod ljudi što ih čini odličnim kandidatima za razvoj lijekova
Analiza miRNA domaćina i herpes simpleks virusa 1 tijekom produktivne i latentne infekcije
No full textViruses significantly perturb cell metabolism during infection and guide
their functions toward efficient virus replication. Previous studies have shown that
herpes simplex virus 1, while also encoding its own microRNAs (miRNAs), leads
to significant changes in host miRNA expression. However, the exact role of such
deregulation has not been elucidated. In this study, we comprehensively
analyzed host and HSV-1 encoded miRNAs during productive and latent infection
by applying a next-generation sequencing approach and bioinformatic analyses
followed by functional studies.
Firstly, to study host miRNA changes during productive HSV-1 infection,
we determined differentially expressed miRNAs in various HSV-1 infected cells
in culture. We found a cluster of co-expressed host miRNAs, miR-183/96/182,
that was reproducibly upregulated in primary cells. Interestingly, we found that
miRNAs of this cluster share common targets, including the Forkhead box O
(FoxO) family of transcription factors. We observed a slight increase followed by
a decrease of FoxO1 and FoxO3 transcripts and proteins during HSV-1 infection,
which coincided with the expression of the miR-183/96/182. However, we found
that overexpression of the miR-183/96/182 cluster is not required for the depletion
of FoxO proteins. Nonetheless, to further examine the roles of these proteins in
infection, we generated cells deficient for FoxO1 and FoxO3 using the CRISPRCas9 technology. Our results show that individual FoxO1 or FoxO3 protein is not
required for efficient virus infection.
In the second part of the study, we analyzed HSV-1 miRNAs in great detail
by comparing a large number of sequencing experiments (i.e., datasets). One
part of the datasets was generated in-house by sequencing, and the other was
obtained from public databases. Thus far, 29 mature miRNAs have been found
encoded by HSV-1, the functions of which are largely unknown. However, we
have noticed significant discrepancies between reports including a lack of
consistency in miRNA sequences.Tijekom infekcije virusi značajno mijenjaju biologiju stanice, te pritom
podređuju i usmjeravaju njezine funkcije u smjeru efikasne virusne replikacije.
Prethodne studije su pokazale da herpes simpleks virus 1, osim što kodira svoje
mikroRNA (miRNA), dovodi i do značajnih promjena u ekspresiji miRNA
domaćina, za što se smatra da bi moglo dovoditi do stvaranja pogodnih uvjeta za
replikaciju virusa, no točne uloge tih promjena nisu detaljno analizirane. Kako bi
opširnije prikazali analizu miRNA domaćina te HSV-1, istraživanje je podijeljeno
na dva dijela, prvi dio, koji čini analiza diferencijalnih promjena miRNA domaćina
tijekom infekcije te drugi dio gdje su detaljnije analizirane miRNA HSV-1.
Prvo, kako bi proučili promjene miRNA domaćina tijekom infekcije HSV-1,
napravili smo profil diferencijalno eksprimiranih miRNA, te pokazali da je klaster
miRNA domaćina koje se zajedno eksprimiraju, miR-183/96/182, reproducibilno
povećano eksprimiran u primarnim stanicama u kulturi. Traženjem zajedničkih
meta navedenih miRNA, od potencijalnih kandidata izdvojili smo Forkhead box O
(FoxO) porodicu transkripcijskih faktora te uočili njihovo blago povećanje nakon
kojeg slijedi opadanje razine transkripata te proteina tokom infekcije HSV-1. Kako
bi ispitali ulogu FoxO u infekciji, metodom CRISPR-Cas9 generirali smo stanice
deficijentne za FoxO1 i FoxO3, no, uspoređujući replikaciju, uočili smo da FoxO1
ili FoxO3 ne igraju značajnu ulogu u replikaciji HSV-1.
Drugo, kako bi detaljnije analizirali miRNA HSV-1, generirali smo i prikupili
setove podataka dobivene sekvenciranjem malih RNA molekula uzoraka
inficiranih sa HSV-1. Istraživanja su do sada pokazala da HSV-1 kodira 29 zrelih
miRNA, čija funkcija nije u potpunosti razjašnjena. U tim dosadašnjim otkrićima
sudjelovale su različite istraživačke grupe koje su u svojim istraživanjima
primjenjivale različite kriterije za otkrića novih miRNA što je na kraju rezultiralo
brojnim odstupanjima, uključujući i točne sekvence miRNA HSV-1. Kako bismo
odredili točne sekvence miRNA HSV-1, te ispitali ekspresiju miRNA u latenciji u
uzorcima čovjeka, usporedili smo sekvence miRNA između različitih virusnih
sojeva i kliničkih izolata. Opsežna bioinformatička analiza pokazala je odstupanja
sekvenci miRNA HSV-1 od objavljenih referentnih sekvenci, čime smo definirali najdominantnije sekvence 29 zrelih miRNA molekula HSV-1 u različitim fazama
HSV-1 infekcije kako bi doprinijeli razumijevanju i olakšali buduće funkcionalne
analize ovih miRNA molekula. Također smo kategorizirali sve poznate miRNA
HSV-1 te sugeriramo da neke od prije objavljenih miRNA možda nisu izvorne
regulatorne molekule, već artefakti sekvenciranja. Nadalje, opisujemo značajno
posttranskripcijsko uređivanje hsv1-miR-H2-3p u latentno inficiranim ganglijima
čovjeka, za razliku od in vitro uzoraka u produktivnoj fazi infekcije. Ovi rezultati
ukazuju na to da virus koristi stanične procese kako bi proširio repertoar mogućih
meta miRNA ili na taj način utječe na njihovu stabilnost
Can Resveratrol Influence the Activity of 11β- Hydroxysteroid Dehydrogenase Type 1? A Combined In Silico and In Vivo Study
No full textThe enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD-1) is an NADPH-dependent reductase, responsible for the activation of cortisol by reducing cortisone. Resveratrol (RES), a type of natural polyphenol, is reported to be able to slow the progression of cancer and cardiovascular disease and improve the health of mice on a high- calorie diet. In this article, we applied molecular docking and molecular dynamics simulations to investigate the possibility of binding RES to 11β-HSD-1. The 11β-HSD-1:RES complex is stable on the μs time scale, and backbone RMSD-based clustering identified three conformations. Special attention was paid to the interaction pattern between the ligand and the target molecule, revealing hydrogen bonds between the hydroxyl group of RES and Thr124, as well as hydrophobic interactions responsible for the binding. In vivo studies demonstrated the ability of resveratrol at a dose of 40 mg/kg to reduce 11β-HSD-1 activity in the liver of rats under conditions of experimental post-traumatic stress disorder (PTSD), as well as in non-stressed animals. In both cases, the resveratrol-induced reduction in 11β-HSD-1 activity was accompanied by an increase in plasma corticosterone levels and a decrease in anxiety levels in the plus maze test
Ekspresija i pročišćavanje receptora mitofagije BNIP3L/NIX
No full textHomeostasis is one of the most important mechanisms that a living organism can have. It maintains and regulates the internal environment of organisms through numerous pathways. One of them is autophagy, a mechanism that balances the production and destruction of cellular organelles and components to preserve cellular integrity. A specific type of autophagy that degrades damaged and dysfunctional mitochondria is called mitophagy. There are a few types of mitophagy, but one of the most interesting ones is the BNIP3L/NIX-mediated mitophagy. The major mediator in this process is BNIP3L/NIX, a protein that comes in a monomeric and dimeric form. The BNIP3L/NIX is positioned on the outer mitochondrial membrane and is crucial for interactions with the autophagosomes and thus promoting mitophagy. To define and study the mechanisms behind this pathway, it would be of great benefit to produce the protein in vitro. As it is a transmembrane protein, this is very challenging. By using pGEX-4T1 plasmids, we successfully achieved the expression of GST-tagged BNIP3L/NIX within E. coli BL21 bacterial strain. Furthermore, we effectively purified the expressed protein utilizing Glutathione Sepharose beads. We have concluded that BNIP3L/NIX proteins can be induced in BL21 bacteria in a 3 hour incubation at 37°C and that they have to be extracted from the bacterial pellet rather than the supernatant. We have also concluded that the GST-NIX wt variant is the hardest to produce and purify as opposed to the mutated GST-NIX G203A and ΔTM variants. These results could potentially lead to new discoveries and research done regarding BNIP3L/NIX and its role in BNIP3L/NIX-mediated mitophagy.Homeostaza je jedan od najvažnijih mehanizama koje živo biće može imati. Ona održava i regulira unutarnji okoliš organizama kroz bezbroj bioloških procesa. Jedan od njih je autofagija, mehanizam koji balansira stvaranje i razgradnju staničnih organela i komponenata kako bi se održao stanični integritet. Posebna vrsta autofagije koja razgrađuje oštećene i disfunkcionalne mitohondrije zove se mitofagija. Postoji nekoliko tipova mitofagije, no jedan od najzanimljivijih je mitofagija posredovana BNIP3L/NIX receptorom. Glavni posrednik ovog procesa je BNIP3L/NIX, protein koji se javlja u obliku monomera i dimera. BNIP3L/NIX se nalazi na vanjskoj membrani mitohondrija te je vrlo važan za interakcije s autofagosomima i pokretanje mitofagije. Kako bi proučili i definirali mehanizme iza ovog procesa, potrebno je proizvesti protein u in vitro uvjetima. BNIP3L/NIX je usidren u vanjsku mitohondrijsku membranu, stoga je njegovo pročišćavanje zahtjevan proces. Koristeći plazmide pGEX-4T1, uspjeli smo ne samo proizvesti GST-BNIP3L/NIX proteine( divlji tip i mutante transmembranske domene) u BL21 bakterijama već i pročistiti ga s glutation sefaroznim kuglicama. Došli smo do zaključka da se BNIP3L/NIX proteini mogu inducirati u BL21 bakterijama uz inkubaciju od 3 sata na 37°C te da se moraju izolirati iz bakterijskog peleta, a ne supernatanta. Također smo zaključili da je GST-BNIP3L/NIX divlji tip varijantu puno teže proizvesti i pročistiti u odnosu na mutirane varijante GST-BNIP3L/NIX G203A i ΔTM (mutante koje ne stvaraju dimere). Ovi rezultati mogu potencijalno dovesti do novih otkrića i istraživanja na BNIP3L/NIX-u i mitofagiji posredovanom BNIP3L/NIX-om
Antitumor effect of the essential oil of the mastic tree (Pistacia lentiscus L.) on breast cancer cell lines
No full textCilj istraživanja: Cilj ovog istraživanja je ispitati potencijalno citotoksično i antitumorsko djelovanje eteričnog ulja izoliranog iz lista tršlje (Pistacia lentiscus L.). na dvije humane karcinomske stanične linije dojke – MDA-MB-231 i MCF-7. Pretpostavljeno je da će se broj karcinomskih stanica smanjiti nakon izlaganja određenim koncentracijama eteričnog ulja tršlje u usporedbi s kontrolnom skupinom.
Materijali i metode: Kako bi izmjerili postotak preživjelih stanica tj. staničnu metaboličku aktivnost, korišten je neradioaktivni kolorimetrijski MTT testni sustav koji se zasniva na redukciji žute tetrazolijeve soli u ljubičaste kristale formazana pomoću metabolički aktivnih stanica. Eterična ulja su se inkubirala u rasponu koncentracija od 1 do 500 µg/mL tijekom 72 sata u tri replikata, a apsorbancija se mjerila na 570 nm na čitaču mikropločica nakon 24, 48 i 72 sata.
Rezultati: Rezultati ovog istraživanja nakon izlaganja karcinomskih stanica različitim koncentracijama eteričnog ulja biljke tršlje se razlikuju za pojedinu karcinomsku staničnu liniju dojke. Najznačajniji rezultati djelovanja na MDA-MB-231 staničnu liniju su nakon inkubacije od 48 sati pri koncentraciji od 500 µg/ml gdje se stanična viabilnost smanjila na 19 % te pri koncentraciji od 250 µg/ml nakon 24 sata gdje se stanična viabilnost smanjila na 52 %. Kod stanične linije MCF-7 najznačajniji rezultati su nakon inkubacije od 48 sati pri koncentraciji od 500 µg/ml gdje se stanična viabilnost smanjila na 30 % te pri koncentraciji od 500 µg/ml nakon 24 sata gdje se stanična viabilnost smanjila na 36 %.
Zaključak: Temeljem dobivenih rezultata ovog istraživanja zaključujemo da eterično ulje lista tršlje ima potencijal u liječenju karcinoma dojke te da u određenom postotku ubija karcinomske stanice. U odnosu na staničnu liniju MDA-MB-231, eterično ulje lista tršlje pokazuje slabije citotoksično djelovanje na staničnu liniju MCF-7. IC50 vrijednost je postignuta za obe stanične linije te se ona smanjuje s povećanjem trajanja inkubacije. Vrijednost je niža kod MDA-MB-231 stanične linije u odnosu na MCF-7 staničnu liniju.Aim of the study: The aim of this research is to examine the potential cytotoxic and antitumor effect of the essential oil isolated from the leaves of the Pistacia lentiscus L. plant on two human breast cancer cell lines - MDA-MB-231 and MCF-7. It was hypothesized that the number of cancer cells will decrease after exposure to certain concentrations of essential oil of mastic tree compared to the control group.
Materials and methods: In order to measure the percentage of surviving cells, i.e. cellular metabolic activity, a non-radioactive colorimetric MTT test system was used, which is based on the reduction of yellow tetrazolium salt into purple formazan crystals by metabolically active cells. Essential oils were incubated in the concentration range from 1 to 500 µg/mL for 72 hours in three replicates, and the absorbance was measured at 570 nm on a microplate reader after 24, 48 and 72 hours. Results: The results of this research after exposure of cancer cells to different concentrations of the essential oil of the mastic tree differ for each breast cancer cell line. The most significant results of the effect on the MDA-MB-231 cell line are after incubation for 48 hours at a concentration of 500 µg/ml where cell viability decreased to 19% and at a concentration of 250 µg/ml after 24 hours where cell viability decreased to 52 %. In the case of the MCF-7 cell line, the most significant results were obtained after incubation for 48 hours at a concentration of 500 µg/ml, where cell viability decreased to 30%, and at a concentration of 500 µg/ml after 24 hours, where cell viability decreased to 36%.
Conclusion: Based on the results of this research, we conclude that the essential oil of the Pistacia Lentiscus L. leaf has potential in the treatment of breast cancer and that it kills cancer cells in a certain percentage. Compared to the MDA-MB-231 cell line, mastic tree leaf extracts show a weaker cytotoxic effect on the MCF-7 cell line. The IC50 value was reached for both cell lines and it decreases with increasing incubation time. The value is lower in MDA-MB-231 cell line compared to MCF-7 cell line
Quillaja saponins as adjuvants
No full textSaponini su biološki aktivne tvari izolirane iz Quillaja saponaria Molina i Quillaja brasiliensis i sposobni su povećati imunogeničnost antigena u antigen-adjuvant kombinaciji. Adjuvantske sposobnosti saponina prvi puta je primijetio francuski veterinar Gaston Ramon, 1925. godine kada je induciran humoralni odgovor protiv difterije i tetanusa. Daljnji istraživački napori doveli su do otkrivanja palete kemijskih struktura saponina. QS-21 izoliran iz Quil-A, primarnog ekstrakta kore Q. saponaria, odabran je za kliničku uporabu na temelju fizikalno-kemijskih svojstava i pripadajućih terapeutskih učinaka. Iako farmakofor saponina nije definiran, postuliranim teorijama predlaže se mehanizam djelovanja. Receptor-posredovani mehanizam nalaže saponin-CD2 receptorsku interakciju na T limfocitima dok receptor-neovisna kolesterol-posredujuća internalizacija i dalje nije odbačena. U svakom slučaju, QS-21 povećava broj antigen-preuzetih antigen prezentirajućih stanica (APC) i potenciranom unakrsnom prezentacijom uzrokuje robusniji Th1/Th2 miješani imunološki odgovor. Osim adjuvantskih, saponini posjeduju antibakterijske, antivirusne i antitumorske sposobnosti po kojima su također zanimljivi u istraživanju cjepiva. U pokušajima otkrivanja farmakofora, šećerni motivi su se pokazali relevantnima u procesima receptorskog podraživanja i molekulske mimikrije, aldehidna C-23 pozicionirana skupina ključna u receptor-posredovanom mehanizmu, a hidrofobni aglikon u kolesterol-ovisnoj internalizaciji. Studije koje otkrivaju utjecaj strukture na aktivnost (SAR studije) nalažu da je cjeloviti kemijski profil saponina, a ne prisutnost ili odsutnost određene funkcionalne grupe zapravo farmakoforski obilježeno. Također, sintezom alternativnih saponina pokušava se poboljšati profil toksičnosti i aktivnosti QS-21, trenutno jedinog saponinskog adjuvanta prisutnog u odobrenim cjepivima. Povećanje stabilnosti i smanjenje hemolitičke aktivnosti QS-21 postiže se njegovim umetanjem u imunostimulirajuće adjuvantne komplekse (ISCOM). U dizajnu učinkovitog cjepiva presuđujuća je antigen-adjuvant odabrana kombinacija. Tako je za bolesti čiji uzročnici unutarstanično parazitiraju, potreban miješani Th1/Th2 odgovor uz Th1 i CTL citotoksično predvođenje. U prilog tome, dokazano je da robusniji imunološki odgovor pokreću adjuvantske kombinacije (AS) u usporedbi sa samostalnim adjuvantom. AS01 i AS15 liposom-bazirane te AS02 emulzija-bazirana formulacija su najintenzivnije predklinički ispitane. Jedini je kliničku upotrebu ostvario AS01 s optimalnim rizik/korist omjerom u cjepivima protiv malarije i herpes zoster.Saponins are biologically active molecules isolated from Quillaja saponaria Molina and Quillaja brasiliensis and are capable of increasing the immunogenicity of antigens in an antigen-adjuvant combination. Saponin adjuvant capabilities were first presented in the 1925 by French veterinarian Gaston Ramon when humoral response was induced while working on diphtheria and tetanus vaccines. Further investigations succeded in reviling the whole palette of chemically different saponins. QS-21 isolated from Quil-A, the primary bark extract of Q.saponaria, is chosen for clinical use based on its physical and chemical uniqueness and belonging therapeutic effects. Despite saponin farmakofor being unknown, there are a few postulated theories that suggest mechanisms of action. Receptor-dependent mechanism proposes saponin interaction with CD2 receptors present on T lymphocytes while receptor-independent, cholesterol-associated internalization still hasn't been rejected. Either way, QS-21 increases the number of antigen-loaded antigen presenting cells (APCs), makes cross-presentation more robust while inducing a mixed Th1/Th2 immune response. Saponins are vaccinologically usable for their antibacterial, antiviral and antitumor properties as well. In attempts for farmakofor reveal, sugar moieties are considered biologically relevant in processes such as receptor stimulation and molecular mimicry of pathogen-associated molecular patterns (PAMPs), C-23 positioned aldehyde group crucial for receptor-associated mechanism and hydrofobic aglicone essential for cholesterol-associated internalization. Structure and activity studies (SAR) are aligned in the opinion that chemical completeness, not the presence or absence of certain functional group, is farmakofor creditable. Also, the synthesis of alternative analogs is needed for the improvement of the toxicity/activity ratio of QS-21, currently the only saponin adjuvant in approved vaccines. With saponin incorporation in immunostimulatory complexes (ISCOM) increasment in stability and decreasment in hemolytic activity is reached. In efficient vaccine design, antigen-adjuvant combination is decisive. Thus, in battle with diseases whose pathogen is intracellular, dominant Th1/Th2 and CTL cytotoxic immune responce is necessary. It is proved that more robust immune response have adjuvant combinations in comparison with adjuvants alone. AS01 and AS15 liposome-based and AS02 emulsion-based formulations are the most intensely studied among all. AS01 is the only one that found its usage in clinical practice because of a suitable risk/benefit ratio for dealing with diseases such as malaria and herpes zoster
Disorders in ribosome synthesis and cancer
No full textRibosomi sintetiziraju proteine. Sastoje se od 80 ribosomskih proteina i 4 rRNA molekula. Poznato je da poremećaji strukture i funkcije ribosoma uzrokuju bolesti ribosomopatije koje imaju predispoziciju za nastanak brojnih vrsta zloćudnih tumora. Međutim, uloga mutacija ribosomskih proteina u nastanku ribosomopatija u ljudi slabo je istražena. Heterozigotne mutacije nekoliko specifičnih ribosomskih proteina ometaju funkciju ribosoma u sintezi proteina, ali i aktiviraju supresor tumora p53. Osim toga, povećanje broja sintetiziranih ribosoma uslijed aktivacije onkogena ili gubitka supresora tumora ima ključnu ulogu u zloćudnoj preobrazbi stanice. Precizni mehanizmi putem kojih navedeni poremećaji dovode do zloćudne preobrazbe do sada nisu detaljno razjašnjeni. U ovom radu prikazano je kako kvalitativne i kvantitativne promjene u sintezi ribosoma dovode do nastanka ribosomopatija. Međutim patogeneza tih bolesti slabo je razjašnjena kao i mehanizmi koji dovode do zloćudne preobrazbe. Od velike je važnosti otkriti putem kojih molekularnih mehanizama dolazi do zloćudne preobrazbe stanica kako bi se otkrile potencijalne mete u liječenju poremećaja u sintezi ribosoma.Ribosomes are composed of 4 rRNAs and 80 distinct ribosomal proteins and they are involved in protein synthesis. It is known that disorders in ribosome structure and functions cause ribosomopathies which have predisposition to develop many types of cancer. However, role of ribosomal protein mutations in the development of ribosomopathies in humans remain largely unknown. Heterozygous mutations in some specific ribosomal proteins disrupt ribosome function in protein synthesis and also activate tumour suppressor p53. Furthermore, increase in the number of ribosomes due to activation oncogene or loss of tumour suppressors plays a key role in malignant cell transformation. Precise mechanisms by which these disorders lead to malignant transformation have not yet been elucidated. In this work it is shown how qualitative and quantitative changes in ribosome synthesis lead to the development of ribosomopathies. However, pathogenesis of these diseases is poorly understood as well as the mechanisms leading to malignant transformation. It is important to find out through which molecular mechanisms the transformation of malignant cells occurs to identify potential targets in the treatment of ribosome synthesis disorders
Diferencijacija akutnog i kroničnog izlaganja METH-u na akumulaciju fAGEs te bihevioralni fenotip
No full textMethamphetamine (METH) and cocaine (COC) are often used recreational
drugs that target dopaminergic synaptic transmission. Repeated exposure
to a specific drug that causes increased motor-stimulant response is termed
behavioral sensitization. In D. melanogaster, behavioral sensitization can
be measured and induced with METH and COC. Dopamine has a role in
changes included in neural plasticity after drug intake, such as behavioral
sensitization. Furthermore, it determines various behaviors, including
amount of sleep and locomotor activity. It increases levels of oxidative
stress and affects accumulation of fluorescent advanced glycation end
products (fAGEs). Based on these findings, we were interested to explore
how different duration of feeding with METH affects motor activating effects
of volatilized METH (vMETH) and volatilized cocaine (vCOC), amount of
locomotor activity and sleep, and biochemical indicators of oxidative stress
such as fAGEs and hydrogen peroxide (H2O2). Feeding with METH did not
influence changes in locomotor activity after vCOC and vMETH
administration using FlyBong. However, we did observe that locomotor
activity and response to psychostimulants decreases with age. Furthermore,
results suggest that neural changes caused by METH are governed by
different mechanisms to those that control neural changes influenced by
COC. Flies pretreated with METH exhibited decreased activity and increased
sleep through the period of fourteen days. Furthermore, we measured levels
of fAGEs and H2O2 in the head of flies. Measurements showed decrease of
H2O2 and increase of fAGEs through the period of fourteen days. This
indicates possible activation of antioxidative defense which decreased H2O2
levels. Increase of fAGEs through fourteen days confirmed its role as
indicators of ageing in D. melanogaster. Further studies are needed to
elucidate the exact effect of oral METH feeding on behavioral and
biochemical factors in D. melanogaster.Metamfetamin (METH) i kokain (COC) su često konzumirane rekreativne
droge, čiji mehanizam utječe na sinaptičku transmisiju dopamina.
Uzastopno izlaganje specifičnoj drogi uzrokuje povećani motorički
stimulirani odgovor kojeg se naziva bihevioralna senzitacija. METH i COC
mogu inducirati bihevioralnu senzitaciju kod D. melanogaster. Dopamin ima
ulogu u promjenama vezane uz neuralnu plastičnost, kao što je bihevioralna
senzitacija, koje nastupaju nakon uzimanja droge. Također, ima ulogu u
regulaciji količine spavanja, lokomotorne aktivnosti te utječe na
akumulaciju fAGEs-a i količinu oksidativnog stresa. Uzevši u obzir prethodne
činjenice, u ovome radu smo željeli istražiti kako različita dužina oralnog
hranjenja METH-om utječe na motorno-aktivirajuće efekte volatiliziranog
METH-a (vMETH) i volatiliziranog kokaina (vCOC), količinu lokomotorne
aktivnosti i spavanja, te biokemijske indikatore oksidativnog stresa fAGEs i
vodikovog peroksida (H2O2). Hranjenje METH-om nije utjecalo na promjene
u lokomotornoj aktivnosti nakon administracije vCOC i vMETH putem
FlyBong platforme. Međutim, uočili smo da lokomotorna aktivnost i odgovor
mušica na psihostimulante opadaju sa starosti. Rezultati sugeriraju da su
neurološke promjene koje uzrokuje METH regulirane mehanizmom koji nije
povezan s mehanizmom koji kontrolira neurološke promjene nakon
administracije COC-a. Nadalje, mušice koje su hranjenje METH hranom
pokazale su smanjenu aktivnost i povećano spavanje kroz četrnaest dana.
Nadalje, mjerili smo koncentraciju fAGEs-a i H2O2 u glavama mušica.
Mjerenja su pokazala opadanje koncentracije H2O2 i porast koncentracije
fAGEs-a kroz četrnaest dana. Pad u koncentraciji H2O2 možemo opravdati
aktivacijom antioksidativne obrane organizma nakon izlaganja METH-u.
Povećanje koncentracije fAGEs-a daje potvrdu prethodnim istraživanjima
koja sugeriraju da je fAGEs indikator starosti mušica. Potrebno je još studija
da bi se saznao točan efekt oralnog hranjenja METH-om na bihevioralne i
biokemijske faktore kod D. melanogaster