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Istraživanje učinka trenutnih analgetika na metabolizam anastrozola
No full textThis research aimed to determine in vitro potential drug-drug interactions between anastrozole and commonly used analgesic drugs such as codeine and/or APAP, individually or in combination. Moreover, an additional goal in vivo was to clarify the potential impact of codeine and/or APAP on the metabolic pathway of anastrozole and, therefore, on anastrozole activity. Up to 80% of the diagnosed breast cancers express oestrogen receptors. Following their activation by oestrogens, oestrogen receptors induce proliferation of the tumour cells, therefore increasing cancer growth. Anastrozole is a third-generation aromatase inhibitor acting on the aromatase (CYP19A1), a key enzyme of the biosynthesis of oestrogens that is highly expressed by breast cancer cells. Paracetamol is the most used over-the-counter medication used either in single-compound form for mild pain or in multi-compound form as with an opioid for moderate to severe pain. Predominantly, paracetamol is combined with codeine. Codeine is a pro-drug, leading to morphine and its metabolites (morphine-3-glucuronide and morphine 6-glucuronide). Morphine acts on the mu-opioid receptors to produce analgesia. Mice livers were used to generate microsomes to determine the production of the anastrozole metabolite, hydroxy-anastrozole and impact of analgesic drugs on anastrozole metabolism in vitro using IC50 assay. For the in vivo experiments, female CB57BL/6J mice were treated with anastrozole (control condition) and codeine, paracetamol, or a combination of both in a single dose (acute treatment) or repeated injections over one week (chronic treatment). Both in vitro and blood samples were analysed and quantified by the liquid chromatography coupled to mass spectrometry (LCMS/ MS) using an isotope dilution method. In vitro, codeine and APAP showed an inhibitory effect on hydroxyanastrozole production in a dose-dependent matter. In vivo, all chronic treatments had a higher ratio of metabolite/parent drug than in the acute Page | 6 treatment, suggesting higher production of the hydroxy-anastrozole. Furthermore, differences between treatments of the same condition indicated that the combination of analgesic drugs and anastrozole had a lower metabolite/parent ratio, suggesting higher alternative metabolic pathways. These findings suggest that codeine and paracetamol impact anastrozole metabolism in mice and, therefore, could potentially impact the metabolism in humans. Further studies in humans are essential to assess the impact of potential interactions between breast cancer endocrine therapy and analgesic treatment with reducing prospective limitations.Svrha ovog istraživanja bila je utvrditi potencijalne interakcije između anastrozola, kodeina i paracetamola, pojedinačno ili u kombinaciji. Štoviše, dodatni cilj ovog istraživanja bio je razjasniti potencijalni utjecaj na metabolički put anastrozola, a time i na samu njegovu aktivnost. Do 80% dijagnosticiranih karcinoma dojke izražava estrogenske receptore, oviseći o estrogenu za proliferaciju. Anastrozol je inhibitor aromataze treće generacije koji djeluje na enzim aromatazu (CYP19A1). Aromataza je ključni enzim bio-sinteze estrogena koji je visoko izražen u stanicama karcinoma dojke. Paracetamol je najčešće korišten lijek dostupan bez recepta koji se koristi ili u obliku jednog spoja za blagu bol ili u obliku više spojeva za umjerenu do jaku bol. Pretežno, paracetamol se kombinira s kodeinom. Kodein je opijat i pro-lijek, koji dovodi do morfija i njegovih metabolita (morfij-3-glukuronid i morfij 6-glukuronid). Morfij djeluje na mu-opioidne receptore kako bi proizveo analgeziju. Jetre miševa korištene su za stvaranje mikrosoma za određivanje proizvodnje metabolita anastrozola, hidroksi-anastrozola i utjecaja analgetskih lijekova na metabolizam anastrozola in vitro s IC50 esejom. Za in vivo eksperimente, ženski CB57BL/6J miševi tretirani su anastrozolom (kontrolno stanje) i kodeinom, paracetamolom ili kombinacijom. Tretman je administriran u obliku jedne doze (akutno liječenje) i ponovljenih injekcija tijekom jednog tjedna (kronično liječenje). In vitro i uzorci krvi analizirani su i kvantificirani tekućom kromatografijom spojenom s masenom spektrometrijom (LC-MS/MS) metodom razrjeđivanja izotopa. In vitro, kodein i APAP pokazali su inhibitorni učinak na proizvodnju hidroksi-anastrozola u ovisnosti o dozi. In vivo, svi kronični tretmani imali su veći omjer metabolita/izvornog lijeka nego u akutnom liječenju. Ovaj rezultat upućuje na veću proizvodnju hidroksi-anastrozola. Nadalje, razlike između tretmana istog stanja pokazale su da kombinacija analgetskih Page | 8 lijekova i anastrozola ima niži omjer metabolita/izvornog lijeka, što potencijalno upućuje na alternativne metaboličke putove. Dobiveni rezultati sugeriraju da kodein i paracetamol utječu na metabolizam anastrozola kod miševa i stoga mogu potencijalno utjecati na metabolizam kod ljudi. Daljnja istraživanja na ljudima su neophodna za procjenu utjecaja potencijalnih interakcija između endokrine terapije raka dojke i analgetskog liječenja uz smanjenje potencijalnih ograničenja
Novel Combination Treatments for BRAFV600E Mutant Colon Cancer with Acquired Resistance to Vemurafenib
No full textMutacija BRAFV600E, zastupljena u oko 10% svih slučajeva raka debelog crijeva, povezana je sa slabim odgovorom na sistemsku terapiju i lošim preživljenjem pacijenata. Vemurafenib (PLX4032), specifični inhibitor proteina BRAFV600E, pokazao je potencijal u liječenju metastatskog melanoma s BRAFV600E mutacijom, ali isti klinički učinak nije zabilježen prilikom terapije pacijenata s rakom debelog crijeva koji imaju mutaciju BRAFV600E. Razlog tome može biti inicijalno slab odgovor na terapiju te brz razvoj rezistencije na vemurafenib. Stoga je cilj ovog doktorskog rada bio otkriti potencijalne nove terapijske mete i molekularne biljege odgovora i razvoja rezistencije stanica raka debelog crijeva sa mutacijom BRAFV600E na vemurafenib. U tu svrhu proveli smo proteomsko profiliranje praćeno bioinformatičkom analizom razlike izražaja proteina između stanica raka debelog crijeva s mutacijom BRAF/divljim tipom KRAS, divljim tipom BRAF/mutacijom KRAS i stanica koje sadrže divlji tip BRAF/KRAS. Validacija potencijalnih biljega karakterističnih za BRAFV600E mutaciju je bila provedena imunohistokemijskom analizom, kao i in silico analizom pomoću TCGA baze podataka. Obje analize su nam potvrdile značajno povećanje izražaja i aktivnosti nukleofozmina (NPM1) kao molekularne značajke specifične za BRAFV600E mutirani rak debelog crijeva. Farmakološka inhibicija NPM1 je potakla povećanu osjetljivost rezistentnih stanica na terapiju vemurafenibom. Inhibicija NPM1 bila je popraćena smanjenim izražajem i aktivnošću c-Myc te njegovih nizvodnih meta PSPH i RanBP1, uključenih u regulaciju biosinteze serina i duplikacije centrosoma. Kako su razine sfingolipida u stanici usko povezane sa sintezom serina, dodatno smo istražili ulogu sfingolipidnog metabolizma u regulaciji rezistencije na vemurafenib u stanicama raka debelog crijeva s BRAFV600E mutacijom. Otkrili smo značajno povećane razine izražaja aktivnih formi sfingozin kinaza 1 i 2 (SphK1, SphK2) u rezistentnim stanicama praćeno povećanjem razine njihovog produkta, sfingozin-1-fosfata (S1P). Farmakološka inhibicija SphK1 ili SphK2 te dodatak egzogenog ceramida C6 doveli su do povećanja osjetljivosti rezistentnih stanica na vemurafenib.Medutim, kombinirana inhibicija SphK2 i BRAF pomoću ABC294640 i PLX4032 je imala snažan sinergistički anti-proliferativni učinak pri najvećem broju kombinacija sub-toksičnih koncentracija ova dva terapeutika, te su stoga daljnja istraživanja bila provedena upravo na ovoj kombinaciji lijekova. Mehanistička studija provedena na kombiniranoj terapiji PLX4032 i ABC294640 ukazala je na inhibiciju AKT kinaze posredovanu smanjenom razinom S1P te posljedično smanjenje izražaja NPM1 i translacijski kontroliranog tumorskog proteina (TCTP). Zaključno, rezultati naše studije ukazuju na ulogu NPM1 kao potencijalnog biljega rezistencije BRAFV600E mutiranog raka debelog crijeva na vemurafenib. Također predlažemo da kombinirana terapija ABC294640 i PLX4032 predstavlja novi terapijski pristup uspješnom dokidanju rezistencije na BRAF inhibiciju u vemurafenib-rezistentnim stanicama raka debelog crijeva s BRAFV600E mutacijom.
Ključne riječi: rak debelog crijeva, BRAFV600E, PLX4032, vemurafenib, kemorezistencije, nukleofozmin, c-Myc, sfingolipidi, sfingozin-1-fosfat, sfingozin kinaza 2, ABC294640, Opaganib, translacijom kontroliran tumorski proteinBRAFV600E mutations, present in about 10% of all colorectal cancer patients, are associated with poor responses to systemic therapy and low patient survival rates. Vemurafenib (PLX4032) is a specific BRAFV600E mutant inhibitor and has been applied in treatments of patients with metastatic melanoma exhibiting this mutation. However, when treating patients with BRAFV600E mutant colorectal cancer, the same response rate is not reached. One possible explanation is the fast-developing resistance to vemurafenib. The aim of this study was detecting novel potential targets that show correlation with the response and the development of resistance to vemurafenib in colon cancer cells and are therefore potential sites for targeted inhibition as a means of increasing sensitivity of cells to vemurafenib. We conducted proteomic and bioinformatic analyses of differences in proteome signatures between BRAFV600E mutant, KRAS mutant and BRAF/KRAS wild type colon cancer cells. Validation of obtained data was preformed using an immunohistochemistry analysis and an in silico analysis of TCGA data; both confirmed the abundance of nucleophosmin (NPM1) in BRAFV600E mutant colon cancer cells. Pharmacological inhibition of NPM1 increased the sensitivity of resistant cells to vemurafenib, which was related to the decrease in the expression and the activity of c-Myc, which in turn decreased the abundance of PSPH and RanBP1, both important for centrosome duplication and serine biosynthesis. Furthermore, we explored the role of sphingolipid metabolism in the regulation of resistance to vemurafenib in BRAFV600E mutated colon cancer cells. Sphingolipids are bioactive lipids whose role in mediating resistance to various therapeutics in colon cancer has already been proven. We detected increased activities of both sphingosine kinases (SphK1 and SphK2) in resistant cells, increased abundance of their product, sphingosine-1-phosphate (S1P), as well as changes in the ceramide metabolism. Pharmacological inhibition of either SphK1 or SphK2, as well as the addition of exogenous ceramide C6, increased sensitivity of resistant cells to vemurafenib. We presume that the inhibition of SphK2 via ABC294640-initiated decrease in AKT activity is due to decreased levels of S1P. In addition, the decrease in AKT activity was followed by the decrease in the translation and the activity of both NPM1 and translationally controlled tumour protein (TCTP). Results from our study suggest that the NPM1/c-Myc axis can be considered a potential therapeutic target to successfully evade resistance to vemurafenib in colon cancer harbouring BRAFV600E. Finally, we provided an insight into how a combined therapy of ABC294640 and PLX4032 can be a novel approach to successfully overcoming the resistance in BRAFV600E mutated colon cancer cells to specific BRAF inhibitor.
Keywords: colon cancer, BRAFV600E, PLX4032, vemurafenib, chemoresistance, nucleophosmin, c-Myc, sphingolipids, sphingosine-1-phosphate, sphingosine kinase 2, ABC294640, Opaganib, translationally controlled tumour protei
Protein groups R01 to R06
No full textThis dataset contains data used in research where we identified the proteins that are differentially distributed in opossum spinal tissue that can and cannot regenerate after injury. The dataset contains excell worksheet with the list of identified proteins.Nano-HPLC-MS/MS was used to analyze the protein gel bands. Excised protein bands were digested in-gel using trypsin. LC-MS/MS analysis was done on an Easy-nLC 1200 (Thermo Scientific, USA) coupled to a QExactive HF mass spectrometer (Thermo Scientific, USA)
Optimizacija protokola za vizualizaciju izvanstaničnih vezikula iz cerebrospinalne tekućine pacijenata s teškom ozljedom mozga na skenirajućem elektronskom mikroskopu
No full textExtracellular vesicles (EVs) represent different types of membranous nanosized vesicles which are produced and released by all cells in the body. Number of studies analysing EVs and their importance in cell to cell communication is continuously growing. Due to EV’s nano size, gold standard imaging method for their visualisation is electron microscopy (EM), both in scanning and transmission mode. In the literature, considerable variability between their preparation protocols and visualisation methods
was observed so the main goal of this study was to optimise EV preparation protocol for EM. Results show that conductivity of the hydrophilic polycarbonate membrane can be obtained by gold-palladium (Au-Pd) sputtering of its surface but precaution must be taken when choosing the thickness since it affects hydrophilicity of the membrane and the attachment of the sample.
Regarding fixation, resulting images did not show significant difference in EV shape and size when comparing two fixatives, glutaraldehyde and paraformaldehyde, and two fixation times, 1 h at room temperature (1 h/RT) and 24 h at +4 °C (24 h/+4 °C). In contrast, resulting images did show differences in shape and size when comparing critical point dried (CPD) and
air dried EVs indicating that CPD preserves surface structure. Lastly, scanning transmission electron microscopy (STEM) images comparing four contrasting solutions, osmium tetroxide aqueous solution, uranyl acetate (UA), UranyLess
TM, and UA-ZeroTM, show that they stain different structures. Taken together, findings in this thesis reveal the complexity of EV
preparation protocol for EM and indicate that further research is needed for its optimisation, especially regarding fixation and staining.Izvanstanične vezikule (IV) obuhvaćaju različite tipove membranom obavijenih nanovezikula koje proizvode i oslobađaju sve stanice organizma. Broj studija koje analiziraju IV i njhovu važnost u komunikaciji između stanica svakodnevno raste. Budući da su IV nanometarskih dimenzija, elektronska mikroskopija (EM), skenirajuća i transmisijska, smatraju se
zlatnim standardom za njihovu vizualizaciju. S obzirom na uočene varijacije u protokolima pripreme i metodama vizualizacije IV, glavni cilj ove studije bio je optimizirati protokol pripreme IV za EM. Kako bi se hidrofilna polikarbonatna membrana mogla vizualizirati skenirajućim elektronskim mikroskopom (SEM), potrebno ju je prekriti slojem vodljivog materijala, zlato-paladij (Au-Pd). Pritom treba paziti na debljinu nanesenog Au-Pd sloja, s obzirom da Au-Pd utječe na hidrofilnost membrane, a time i na vezanje IV za membranu. Slike IV fiksiranih glutaraldehidom ili paraformaldehidom, 1 h pri sobnoj temperaturi ili 24 h pri +4 °C, ne pokazuju značajnu razliku u obliku i veličini. IV osušene metodom sušenja kritičnom točkom (eng. Critical point drying, CPD) pokazuju razlike u obliku i veličini, u usporedbi s IV osušenima na zraku što ukazuje da CPD omogućuje očuvanje strukture uzorka. Nadalje, slike dobivene SEM-om, uz korištenje detektora za transmitirane elektrone (STEM), ukazuju da četiri kontrastne otopine, vodena otopina osmij tetroksida, uranil acetat (UA),
UranyLessTM, i UA-ZeroTM , kontrastiraju različite strukture. Zaključno, rezultati ove studije otkrivaju kompleksnost protokola pripreme IV za EM i ukazuju na potrebu za daljnjim istraživanjima, posebice fiksacije i kontrastiranja, kako bi se postigla optimizacija protokola
Revealing molecular and cellular mechanisms involved in neuroprotection and neuroregeneration after central nervous system injury
No full textThe inability of adult mammals to regenerate central nervous system (CNS) after injury is one of the greatest challenges of modern neuroscience, which has not yet revealed how to fully restore the lost functions of damaged neuronal tissue in the brain and spinal cord.To address this problem, we established two new experimental in vitro models suitable for CNS injury research: in vitro culture of neonatal rat spinal cord tissue with longer period of viability (more than 24h), in which we investigated molecular and cellular mechanisms of neurodegeneration and neuroprotection, and primary cortical cell cultures derived from
postnatal opossums, to investigate CNS development and the possibility of its regeneration after mechanical or pharmacological experimental injury. The main goal of the research was to discover key cellular and molecular mechanisms that control and enable neuroprotection and neuroregeneration of the mammalian CNS, in order to develop new strategies for treatment of CNS injuries.The results of the study showed that the heat shock protein HSP70 plays a major role in the survival of motoneurons after experimental rat spinal cord injury in vitro and thus has a neuroprotective effect. Therefore, we pharmacologically induced HSP70 expression and confirmed early protection of motoneurons during acute in vitro rat spinal cord injury. Furthermore, the results of the neuroregeneration studies on neonatal opossums indicated that transcription factor ATF3 plays an important role in control of maturation of mammalian neural progenitor stem cells, and that, consequently, ATF3 is an important factor in regeneration of brain neurons of young opossum after injury. The overall achievements of this study could, in future, allow the development of new neuroprotective strategies for the treatment of mammalian brain and spinal cord injuries, as well as new strategies to promote neuroregeneration of mammalian CNS, which lose this ability during early embryonic or postnatal development.Nemogućnost odraslih sisavaca da regeneriraju središnji živčani sustav (SŽS) nakon ozljede jedan je od najvećih izazova suvremene neuroznanosti koja još uvijek nije odgovorila na pitanje kako u potpunosti vratiti funkciju oštećenog živčanog tkiva mozga i leđne moždine. Kako bi pridonijeli rješavanju ovog problema, uspostavili smo dva nova eksperimentalna
in vitro modela pogodna za istraživanje ozljeda SŽS-a: dugotrajnu (više od 24h) in vitro kulturu tkiva leđne moždine neonatalnog štakora na kojoj smo istraživali molekularne i stanične mehanizme neurodegeneracije i neuroprotekcije, te primarne stanične kulture korteksa postnatalnog oposuma koje smo koristili za istraživanje razvoja SŽS, te
mogućnosti njegove regeneracije nakon eksperimentalne mehaničke ili farmakološke ozljede. Glavni cilj istraživanja bio je otkrivanje ključnih staničnih i molekularnih mehanizama koji kontroliraju i omogućuju neuroprotekciju i neuroregeneraciju SŽS-a sisavaca, kako bi se razvile nove strategije liječenja ozljeda istoga. Rezultati istraživanja pokazali su da protein toplinskog šoka HSP70 igra jednu od glavnih uloga u preživljavanju motoneurona nakon eksperimentalne ozljede leđne moždine štakora in vitro te da stoga ima neuroprotektivno djelovanje. Farmakološki smo stoga inducirali ekspresiju HSP70 i potvrdili ranu zaštitu motoneurona tijekom akutne ozljede tkiva leđne moždine štakora in vitro. Nadalje, rezultati istraživanja neuroregeneracije SŽS-a oposuma ukazali su da transkripcijski faktor ATF3 ima važnu ulogu u kontroli sazrijevanja neuralnih matičnih praroditeljskih stanica sisavaca, te posljedično da je važan čimbenik u regeneraciji moždanih neurona mladih oposuma nakon ozljede. Sveukupna postignuća ovog istraživanja mogla bi u budućnosti omogućiti razvoj novih
neuroprotetivnih strategija za tretman ozljeda mozga i leđne moždine sisavaca, kao i novih strategija koje bi potaknule neuroregeneraciju SŽS-a sisavaca, koji tu sposobnost gube tijekom ranog embrionalnog ili postnatalnog razvoja
Protein aggregation of NPAS3, implicated in mental illness, is not limited to the V304I mutation
No full textAn emerging phenomenon in our understanding of the pathophysiology of mental illness is the idea that specific proteins may form insoluble aggregates in the brains of patients, in partial analogy to similar proteinopathies in neurodegenerative diseases. Several proteins have now been detected as forming such aggregates in the brains of patients, including DISC1, dysbindin-1 and TRIOBP-1. Recently, neuronal PAS domain protein 3 (NPAS3), a known genetic risk factor for schizophrenia, was implicated through a V304I point mutation in a family with major mental illness. Investigation of the mutation revealed that it may lead to aggregation of NPAS3. Here we investigated NPAS3 aggregation in insular cortex samples from 40 individuals, by purifying the insoluble fraction of these samples and testing them by Western blotting. Strikingly, full-length NPAS3 was found in the insoluble fraction of 70% of these samples, implying that aggregation is far more widely spread than can be accounted for by this rare mutation. We investigated the possible mechanism of aggregation further in neuroblastoma cells, finding that oxidative stress plays a larger role than the V304I mutation. Finally, we tested to see if NPAS3 aggregation could also be seen in blood serum, as a more accessible tissue than the human brain for future diagnosis. While no indication of NPAS3 aggregation was seen in the serum, soluble NPAS3 was detected, and was more prevalent in patients with schizophrenia than in those with major depressive disorder or controls. Aggregation of NPAS3 therefore appears to be a widespread and multifactorial phenomenon. Further research is now needed to determine whether it is specifically enhanced in schizophrenia or other mental illnesses
Utjecaj cirkadijalne modulacije na oksidativni status Drosophile melanogaster
No full textCircadian rhythm is present in almost all living organisms due to the rotation
of the Earth and regular light and dark changes. Its role is evident in 24
hour modulation of biological processes such as the sleep/wake cycle.
Disrupted circadian rhythm correlates with oxidative stress, which is caused
by increased production of Reactive Oxygen Species (ROS). In this thesis,
we explored the connection between the circadian rhythm and oxidative
state in D. melanogaster. Based on similarity in the responsiveness to
volatilized cocaine between flies with mutation in timeless gene and wild
type flies, we were interested to see whether these similarities will be
paralleled in their redox regulation. An indicator of redox state was the
concentration of the oxidative metabolism byproduct H2O2 in head
homogenates. For detection of H2O2 we used the dihydroethidium (DHE)
staining. Furthermore, we wanted to determine if H2O2 production is
regulated by circadian rhythm. Measurements were conducted in flies
raised in conditions of 12 hours of light-12 hours of dark (LD) and in
conditions of constant dark (DD). We determined that H2O2 production was
regulated by circadian rhythm for wt flies. However, H2O2 production in DD
conditions was increased overall. In tim mutants there was an indication of
circadian regulation of H2O2 production in LD conditions but the rhythm was
abolished in DD conditions. wt and tim flies differed significantly in the 24
hour modulation of H2O2 in DD, but less so in LD. Further studies are needed
to elucidate the role of tim in the regulation of H2O2 amount and the
connection with cocaine responsiveness. Taken together, our data suggest
that redox status is regulated in a circadian fashion and that a functional
tim gene is essential for maintaining circadian rhythmicity.Cirkadijalni ritam je prisutan u skoro svim organizmima. Nastao je kao
posljedica rotacije Zemlje i izmjene dana i noći. Važan je za regulaciju
brojnih bioloških procesa kao što je i ciklus spavanja. Narušeni cirkadijalni
ritam je često povezivan s pojavom oksidativnog stresa koji nastaje kao
posljedica povišene produkcije reaktivnih kisikovih vrsta (ROS). U ovome
radu, istražili smo povezanost cirkadijalnog ritma i oksidativnog statusa u
D.melanogaster. S obzirom da su wild type i timeless mušice pokazale
sličnost u odgovoru na izlaganje volatiliziranom kokainu, zanimalo nas je
hoće li sličnost biti prisutna i kod regulacije redoksa. Indikator redoks
statusa je bila koncentracija H2O2 u homogenatima glava mušica. H2O2 je
nusprodukt oksidativnog metabolizma, a za njegovu detekciju korištena je
DHE metoda bojanja. Nadalje, ispitali smo je li proizvodnja H2O2 regulirana
cirkadijalnim ritmom. Mjerenja su provedena na mušicama koje su držane
u LD ili DD uvjetima. Pokazali smo da je kod wt mušica H2O2 proizvodnja
regulirana cirkadijalnim ritmom. Međutim, proizvodnja H2O2 je bila
sveukupno povišena u DD uvjetima. Kod tim mutanata, indikacije
cirkadijalne modulacije u proizvodnji H2O2 prisutne u LD uvjetima nisu
uočene i u DD uvjetima. wt i tim mušice se značajno razlikuju u modulaciji
H2O2 koncentracija u DD uvjetima dok je u LD uvjetima razlika značajno
manja. Potrebno je još studija koje će odrediti točnu ulogu tim gena u
regulaciji H2O2 i koje će definirati njegovu ulogu u procesu odgovora na
izlaganje kokainu. Sveukupno, dobiveni podatci sugeriraju da je
metabolizam ROS-a povezan s cirkadijalnim ritmom te da je funkcionalni
gen tim esencijalan za održavanje cirkadijalne ritmičnosti
Razvoj inovativnog phage display protokola za probir antitijela za unaprijeđenje rane detekcije tumora
No full textAdvantages such as faster and cheaper production of highly specific engineered antibodies by phage display in vitro method enabled application of these antibodies for diagnostic or treatment purpose for many diseases including cancer. The goal of this master thesis was to develop an innovative phage display protocol for antibody screening, which will be applied for improvement of early cancer detection. Briefly, the developed protocol relies on a linear amplification of a donor’s antibody coding sequences, allowing to prepare a phage bank which is an exact molecular fingerprint of the donor’s antibodies repertoire. This protocol differs from traditional phage display banks preparation which rely on exponential amplification, selecting the most represented sequences and leading to a biased phage bank. Plasmids strands were converted to RNA, needed for the future preparation of the functional phagemid. Sequences were then combined with plasmid's strands, and the resulting phagemid was inserted in bacterias for positive clones screening. The perspective of this work will be to optimize the protocol and construct an antibody phage library with a high divers
Rationality of prescribing and use of antimicrobial drugs
No full textOtkriće antimikrobnih lijekova značajno je unaprijedilo medicinu, ali i čovječanstvo. No zlouporaba antibiotika, prvenstveno u zdravstvene svrhe ljudi, odnosno njihova neracionalna primjena, doprinijela je razvoju antimikrobne rezistencije. Nekada se smatralo da rezistencija bakterija na antibiotike neće biti problem jer će farmaceutska industrija stalno proizvoditi nove antibiotike, no danas znamo da to nije tako i da moramo očuvati djelotvornost dostupnih antibiotika. Povećanje broja multirezistentnih bakterija, uz nedostatak novih antibiotika, globalna je prijetnja javnom zdravstvu. Glavni, iako ne jedini, razlog širenja antimikrobne rezistencije je prekomjerna i neprimjerena uporaba antibiotika u zdravstvene svrhe ljudi. Znanje i savjesnost zdravstvenih djelatnika imaju velik utjecaj na navike propisivanja antibiotika. Dok znanje, stav i uvjerenja opće populacije izravno utječu na navike prilikom korištenja antibiotika. Istraživanje se temelji na anketnom ispitivanju opće populacije i daje uvid propisuju li se i koriste antibiotici u Republici Hrvatskoj racionalno. Postoje značajni nedostaci prilikom propisivanja i korištenja antibiotika. Neki od primjera su nepotrebno propisivanje, posebice za liječenje respiratornih infekcija, koje su najčešće virusne etiologije, zatim propisivanje bez dokaza o uzročniku infekcije temeljenih na mikrobiološkim nalazima. Najčešći nedostaci u korištenju antibiotika su zanemarivanje uzimanja jedne ili više doza antibiotika te prijevremeni prestanak uzimanja terapije. Učestalost gastrointestinalnih nuspojava je mala i opća populacija je upoznata s korisnim učinkom probiotika. Farmaceutski djelatnici, kao izdavači lijekova, imaju veliku ulogu u davanju uputa o korištenju antibiotika i probiotika općoj populaciji. Mali, ali značajan, dio populacije imao je infekciju uzrokovanu rezistentnom bakterijom. Dio navodi da je uzrok tome bilo bolničko liječenje i intravenozna terapija antibiotika, a drugi dio navodi da je infekciji prethodilo češče korištenje antibiotika. Veći dio opće populacije nije upoznat sa značenjem i značajem antimikrobne rezistencije. Potrebno je što prije započeti edukaciju stanovništva, svih starosnih dobi, o nužnosti racionalne uporabe antibiotika i prijetnji antimikrobne rezistencije. Svaki pojedinac, doprinosi rješavanju ovog problema, od zdravstvenih djelatnika do opće populacije. Važno je objasniti stanovništvu da su antibiotici lijekovi koji, kada ih koristi pojedinac, imaju učinak na cijelu zajednicu.The discovery of antimicrobial drugs has significantly improved medicine, but also humanity. But the misuse of antibiotics, primarily for human health purposes, has contributed to the development of antimicrobial resistance. It was thought that bacterial resistance would not be a problem because the pharmaceutical industry would constantly produce new antibiotics, but today we know that this is not the case and that we must preserve the effectiveness of the available antibiotics. The increase in the number of multidrug-resistant bacteria, along with the lack of new antibiotics, is a global threat to public health. The main reason, though not the only, for the spread of antimicrobial resistance is the excessive and inappropriate use of antibiotics for human health purposes. Knowledge and conscientiousness of health professionals have a major impact on antibiotic prescribing. While the knowledge, attitude and beliefs of the general population directly influence habits when using antibiotics. The research is based on a survey of the general population and provides insight into whether antibiotics are prescribed and used rationally in the Republic of Croatia. There are significant drawbacks when prescribing and using antibiotics. Some of the examples are unnecessary prescribing, especially for the treatment of respiratory tract infections, which are, most commonly, of viral etiology. Followed by prescribing of antibiotics without evidence of infectious agents based on microbiological analysis. The most common shortcomings in the use of antibiotics are neglect of taking one or more doses of antibiotics and premature cessation of therapy. The incidence of gastrointestinal side effects is low and the general population is aware of the beneficial effect of probiotics. Pharmacists, as drug dispensers, play a major role in providing instructions on the use of antibiotics and probiotics to the general population. A small but significant part of the population had an infection caused by resistant bacteria. Part states that the cause of this was hospital treatment and intravenous antibiotic therapy, and the other part states that the infection was preceded by more frequent use of antibiotics. The majority of the general population is not familiar with the meaning and significance of antimicrobial resistance. It is necessary to start educating the population, of all ages, as soon as possible about the necessity of rational use of antibiotics and the threat of antimicrobial resistance. Every individual contributes to solving this problem, from health professionals to the general population. It is important to explain to the population that antibiotics are drugs that, when used by an individual, have an effect on the whole community
Primjena nanotehnologija za mjerenje koncentracije i promjera izvanstaničnih vezikula iz likvora čovjeka: usporedba mikroskopije atomskih sila i opažanja pomoću podesivog otpornog pulsa
No full textExtracellular vesicles (EVs) are membranous nanoparticles that are currently intensively studied for their qualitative and quantitative changes in pathophysiological conditions. Namely, EVs are secreted by practically all cells and their molecular composition reflects the type and state of originating cells. However, research of EVs is hampered due to lacking methods for precise and reliable quantification.
The goal of this study was to improve EV size and concentration measurement in their natural environment by applying novel technologies able to directly measure individual nanoparticles. Such quantification is obtained by Tunable Resistive Pulse Sensing (TRPS) and Atomic Force Microscopy (AFM). The size and concentration of EVs were determined in cerebrospinal fluid (CSF) from patients with severe traumatic brain injury, a condition in which EVs have been previously described to change their physical properties.
TRPS measurements of the CSF sample showed a mean EV diameter of 64.3 ± 30.7 nm and a concentration of 1.3 x 10^9 particles/mL. The same CSF sample was used for the AFM measurement for which a novel protocol was introduced in tapping mode to obtain images in a liquid environment. Particle diameter distribution was calculated from 10 1x1 µm images containing 170 nanoparticles, resulting in a mean diameter of 59.2 ± 22.1 nm, and a concentration approximation of 1.7 x 10^10 particles/mL.
Taken together, both nanotechnologies resulted in similar EV diameter values of around 60 nm. Yet, TRPS brings a quantitative advantage in a simultaneous measurement of EV size and concentration. This research will further contribute to the characterisation of quantitative changes of nanoparticles in biofluids.Izvanstanične vezikule (IV) su membranske nanočestice koje se trenutno intenzivno proučavaju zbog njihove kvalitativne i kvantitativne promjene u patofiziološkim stanjima. Naime, IV-e izlučuju praktički sve stanice, a njihov molekularni sastav odražava vrstu i stanje stanica iz kojih potječu. Međutim, istraživanje IV-a je otežano zbog nedostatka metoda za preciznu i pouzdanu kvantifikaciju.
Cilj istraživanja bio je poboljšati mjerenje veličine i koncentracije IV-a u njihovom prirodnom okruženju primjenom novih tehnologija koje mogu izravno mjeriti pojedinačne nanočestice. Takva se kvantifikacija dobiva opažanjem pomoću podesivog otpornog pulsa (engl. Tunable Resistive Pulse Sensing, TRPS) i mikroskopijom atomske sile (engl. Atomic Force Microscopy, AFM). Utvrđena je veličina i koncentracija IV-a iz likvora pacijenata s teškom ozljedom mozga, stanju u kojem su IV-e već opisane kako mijenjaju svoja fizička svojstva.
TRPS mjerenja uzorka likvora pokazala su srednji promjer IV-a od 64,3 ± 30,7 nm i koncentraciju od 1,3 x 10^9 čestica/ml. Isti uzorak likvora korišten je za mjerenje AFM-om za koje je uveden novi protokol u načinu tapkanja kako bi se dobile slike IV-a u tekućem okruženju. Raspodjela promjera čestica izračunata je iz 10 slika veličine 1x1 µm s ukupno 170 nanočestica, što je rezultiralo srednjim promjerom od 59,2 ± 22,1 nm i približnom koncentracijom od 1,7 x 10^10 čestica/ml.
Sveukupno, obje nanotehnologije rezultirale su sličnim vrijednostima promjera IV-a od oko 60 nm. Ipak, TRPS donosi kvantitativnu prednost u istodobnom mjerenju veličine i koncentracije IV-a. Ovo će istraživanje dodatno doprinijeti karakterizaciji kvantitativnih promjena nanočestica u biotekućinama