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Imunološkim sustavom posredovana regulacija metabolizma aminokiselina tijekom virusnih infekcija
No full textIn response to viral infections, the body uses a wide range of immune mechanisms to protect itself from the invading pathogen. Moreover, in a recent decade it has been appreciated that immune cell signals play a significant part in the regulation of the metabolism and that they are implicated in the development of the various metabolic diseases. In addition, recent studies showed that immune-endocrine interactions have a crucial role in the maintenance of the host physiology and defense. However, the interorgan processes by which immune cells communicate with endocrine cells to coordinate endocrine function and thus to modulate amino acid metabolism during viral infections remains largely unclear. Moreover, what is the purpose of these changes remains fundamentally unexplored. Therefore, our hypothesis is that, in order to minimize the risk of excessive tissue damage and to prevent virus replication, the immune system actively modifies the metabolism of amino acids which boots immune response. Indeed, our research revealed that the immune system during viral infections actively modulates amino acids metabolism, and in particular axis of the homocysteine and cysteine levels. Moreover, we showed that targeting this metabolic pathway helps to modulate the immune response and thus control viral infections. Finally, we confirmed that monitoring and manipulating homocysteine/cysteine levels could be a potential strategy for treatment of viral infections. Therefore, the findings of this thesis will help to clarify the specific mechanisms by which homocysteine and cysteine metabolism regulate immune cell function, cytokine production, and viral replication.Kao odgovor na virusne infekcije, tijelo koristi širok spektar imunoloških mehanizama kako bi se zaštitilo od invazivnih patogena. U posljednjem desetljeću prepoznato je da signalizacija imunoloških stanica igra značajnu ulogu u regulaciji metabolizma, te je uključena u razvoj raznih metaboličkih bolesti. Štoviše, nedavne studije pokazale su da imunološko-endokrine interakcije imaju ključnu ulogu u održavanju fiziologije domaćina i obrambenih mehanizama. Međutim, međustanični mehanizmi kojima imunološke stanice komuniciraju s endokrinim stanicama kako bi koordinirale endokrinu funkciju i time modulirale metabolizam aminokiselina tijekom virusnih infekcija, ostaju uglavnom nejasni. Nadalje, svrha ovih promjena ostaje sveobuhvatno neistražena. Stoga je naša hipoteza da imunološki sustav, u cilju smanjenja rizika od prekomjernog oštećenja tkiva i suzbijanja replikacije virusa, aktivno mijenja metabolizam aminokiselina čime se pojačava imunološki odgovor. Naše istraživanje pokazalo je da imunološki sustav tijekom virusnih infekcija aktivno modulira metabolizam aminokiselina, a posebice razine homocisteina i cisteina. Dodatno smo pokazali da ciljanje ovog metaboličkog puta pomaže modulaciji imunosnog odgovora, a time i nadzoru virusne infekcije. Konačno, potvrdili smo da praćenje i manipuliranje razinama homocisteina i cisteina može biti potencijalna strategija za liječenje virusnih infekcija. Rezultati ovog istraživanja pomoći će razjasniti specifične mehanizme putem kojih metabolizam homocisteina i cisteina regulira funkcionalnost imunoloških stanica, proizvodnju citokina i replikaciju virusa
WapAI posreduje kompeticiju kod rojenja i stvaranje biofilma Bacillus subtilis
No full textBacteria are unicellular organisms but nonetheless they engage in multicellular behaviours like swarming motility and biofilm formation. Essential for their survival in such communities are mechanisms responsible for cooperation and competition. Moreover, bacteria are able to discriminate between strains based on the relatedness and treat less related strains differently than those that are genetically identical (clones) or highly related. This behaviour, referred to as kin discrimination, has been recently described for environmental strains of Bacillus subtilis and the tRNase toxin WapA has been identified as a potential kin discrimination locus. However, the mechanisms behind this process are not sufficiently known. Here, we characterize a role of the WapAI toxin-immunity system in competition between isogenic or non-kin strains. We studied their interactions in pellicles and swarming communities by determining their fitness by colony forming units. We also documented spatial distribution of two types of cells in pellicles by confocal microscopy. Our results show that the fitness of the toxin sensitive strain is significantly reduced compared to the toxin producing strain, regardless whether it is competed against parental or non-kin strain during swarming. In pellicles, parental strain did not outcompete the isogenic wapAI mutant, but the fitness of the non-kin mutant was reduced. Interestingly, mutant and parental cells co-aggregated into larger patches while two differentially labelled but otherwise isogenic strains mixed very well. Patchiness was not observed in non-kin combination, where the wild type outcompeted the mutant strain, which was undetectable by fluorescent microscopy. We conclude that competition in swarms is fiercer than in pellicles, probably due to less intense mixing of genotypes in pellicles.Bakterije su jednostanični organizmi koji stupaju u višestanične interakcije kao što su rojenje (engl. swarming) i formacija biofilmova. Za njihovo preživljenje u spomenutim zajednicama su nužni mehanizmi odgovorni za kooperaciju i kompeticiju. Nadalje, bakterije imaju sposobnost diskriminacije (razlikovanja) sojeva na temelju povezanosti gdje manje srodne sojeve tretiraju drugačije od genetički identičnih sojeva (klonovi) ili visoko srodnih. Spomenuto ponašanje se naziva diskriminacija srodnika (engl. kin discrimination), nedavno opisano u okolišnim sojevima bakterije Bacillus subtilis, a toksin WapA (tRNaza) kao potencijalni lokus za navedenu diskriminaciju. Međutim, mehanizmi iza ovog procesa nisu dovoljno znani. U ovom radu smo opisali ulogu WapAI sistema u kompeticiji između izogenih ili nesrodnih sojeva. Proučavali smo interakcije u peliklima i u zajednicama rojenja brojanjem kolonija na ploči (engl. colony forming units, CFU). Istražili smo prostornu distribuciju dvije vrste stanica u peliklima konfokalnom mikroskopijom. Rezultati su pokazali da je sposobnost razmnožavanja soja osjetljivog na toksin značajno smanjena u usporedbi sa sojem koji stvara toksin bez obzira je li u kompeticiji s roditeljskim ili nesrodnim sojem tijekom rojenja. U peliklima, roditeljski soj nije prevladao izogenu wapAI mutantu, ali je frekvencija nesrodne mutante bila značajno reducirana. Također, stanice mutante i roditeljskog soja su agregirale u veće nakupine dok su različito obilježeni, ali izogeni sojevi iskazali vrlo dobro miješanje. Nakupljanje stanica nije bilo zabilježeno u nesrodnim kombinacijama, gdje je divlji tip prevladao mutantu koju nije bilo moguće zapaziti fluorescentnim mikroskopom. Zaključno, kompeticija tijekom rojenja je snažnija nego u peliklima vjerojatno zbog slabijeg miješanja genotipova u peliklima
Preparation and Characterization of Lifitegrast-Loaded Hyalurosomes
No full textLifitegrast je djelatna tvar protuupalnog lijeka Xiidra® koji se upotrebljava za liječenje sindroma suhog oka u obliku oftalmološke otopine. Nedavni literaturni podaci pokazuju kako se lifitegrast razgrađuje na onečišćenja pod utjecajem termolize, kisele i alkalne hidrolize te oksidacije. Kako bi se riješio problem stabilnosti lifitegrasta i poboljšala postojeća formulacija lijeka, cilj je bio uklopiti lifitegrast unutar hijalurosoma te praćenjem stabilnosti (sadržaja) lifitegrasta utvrditi utjecaj hijalurosoma na njegovu stabilnost. Hijalurosomi su nanometrijske vezikule, slične liposomima, koje nastaju povezivanjem hijaluronske kiseline i molekula fosfolipida. Općenito, hijalurosomi su strukture koje mogu uklopiti djelatne tvari, čime doprinose povećanju stabilnosti jer fizički izoliraju lijek od vanjskih uvjeta koji mogu uzrokovati njegovu degradaciju. S ciljem optimizacije pripreme liposoma i hijalurosoma korištene su dvije metode, od kojih je metoda hidratacije, ekološki prihvatljiva metoda bez upotrebe organskih otapala, proizvela najuniformnije vezikule u pogledu veličine, indeksa polidisperznosti i zeta potencijala. Fizikalne karakteristike (veličina vezikula, indeks polidisperznosti i zeta potencijal) hijalurosoma uspoređene su s onima liposoma koji sadrže istu količinu fosfolipida. Dok je stabilnost vezikula ispitivana praćenjem fizikalnih karakteristika tijekom mjesec dana na 5 °C i 25 °C. Pokazano je da su fizikalne karakteristike hijalurosoma ostale stabilne tijekom mjesec dana pri temperaturama od 5 °C i 25 °C, dok su kod liposoma veličina i indeks polidisperznosti ostali stabilni, a zeta potencijal je pokazao tendenciju povećanja, što bi nakon dužeg razdoblja skladištenja moglo dovesti do agregacije liposoma. Studije prisilne razgradnje pokazale su da su formulacije hijalurosoma s uklopljenim lifitegrastom stabilne u pogledu fizikalnih karakteristika i sadržaja lifitegrasta pri 25 °C / 60 % RV i 50 °C / 75 % RV. Međutim, kada je promatran utjecaj temperature i oksidansa na razgradnju lifitegrasta, uočene su promjene u veličini i zeta potencijalu hijalurosoma, pH uzorka te sadržaju lifitegrasta. Formulacije hijalurosoma s većom koncentracijom fosfolipida (60 mg/ml) zadržavaju veći postotak početnog sadržaja lifitegrasta u oba uvjeta skladištenja (25 °C / 60 % RV + H2O2 i 50 °C / 75 % RV + H2O2) u usporedbi s kontrolnim uzorcima. Dobiveni rezultati otvaraju put potencijalnoj upotrebi hijalurosoma za isporuku lifitegrasta.Lifitegrast is the active ingredient in the anti-inflammatory drug Xiidra®, used to treat dry eye syndrome. Recent literature data show that lifitegrast degrades into impurities under the influence of thermolysis, acidic and alkaline hydrolysis, and oxidation. To address the stability issue of lifitegrast and improve the existing drug formulation, the goal was to encapsulate lifitegrast within hyalurosomes and determine the impact of hyalurosomes on its stability. Hyalurosomes are nanometric vesicles, similar to liposomes, formed by the association of hyaluronic acid and phospholipid molecules. Generally, hyalurosomes are structures that can encapsulate active substances, thereby contributing to increased stability by physically isolating the drug from external conditions that may cause degradation. To optimize the preparation of liposomes and hyalurosomes, two methods were used, among which the hydration method, an environmentally friendly method that does not use organic solvents, produced the most uniform vesicles in terms of size, polydispersity index, and zeta potential. The physical characteristics (vesicle size, polydispersity index, and zeta potential) of hyalurosomes were compared with those of liposomes containing the same amount of phospholipids. Meanwhile, the stability of the vesicles was evaluated by monitoring the physical characteristics over a month at 5 °C and 25 °C. It was shown that the physical characteristics of hyalurosomes remained stable at both 5 °C and 25 °C, whereas for liposomes, size and polydispersity index remained stable, but the zeta potential showed a tendency to increase, which could potentially lead to liposome aggregation after a longer storage period. Forced degradation studies have shown that hyalurosome formulations with encapsulated lifitegrast are stable in terms of physical characteristics and lifitegrast content at 25 °C / 60% RH and 50 °C / 75% RH. However, when examining the effect of temperature on the oxidation of lifitegrast, changes were observed in the size and zeta potential of the hyalurosomes, sample pH, and lifitegrast content during storage. Formulations of hyalurosomes with the highest phospholipid concentration (60 mg/ml) retain a higher percentage of the initial lifitegrast content under both storage conditions (25 °C / 60 % RH + H2O2 i 50 °C / 75 % RH + H2O2) compared to the control samples. The obtained results pave the way for the potential use of hyalurosomes for the delivery of lifitegrast
Ex vivo ekspanzija i fenotipska karakterizacija mišjih primarnih T regulatornih stanica
No full textFoxp3+ CD25+ regulatory T cells (Tregs) are a rare subset within the CD4+ T cell compartment, playing a vital role in immune system regulation. Because of their immunosuppressive ability, Tregs are an attractive potential new tool for treating autoimmune diseases and preventing graft rejection. Although the population of Treg cells is well-defined by the intracellular expression of the transcription factor Forkhead box protein 3 (Foxp3), no defined surface marker distinguishes Tregs from other T cells, making reliable phenotypic characterization and isolation challenging. Furthermore, most isolation and expansion protocols are designed for human Tregs, representing a significant drawback since mice are important model organisms in the preclinical phase of drug development. A range of methodologies was employed. Treg cells were isolated from mouse spleens using immunomagnetic separation. Consequently, cells were activated and expanded for 15 days, using different ratios of Treg cells-to-activation beads in the presence of mouse interleukin-2 (mIL-2) cytokine. After the staining for viability, surface, and intracellular markers, the
phenotype of Tregs and other T cell subsets was analyzed using flow cytometry (FC). To investigate the transcriptional profiles of subpopulations within the Tregs samples, we performed the 10x Genomics single-cell RNA sequencing (scRNA-seq). This study resulted in the successful establishment of isolation and expansion protocols for mouse Foxp3+ CD25+ Tregs, as well as comprehensive FC staining panels. The phenotype and transcriptome analysis revealed the heterogeneity of the Treg population already upon isolation from secondary lymphatic tissue. ScRNA-seq analysis provided a deeper insight into the Treg cells' transcriptome and revealed other important biomarkers for contaminants-free isolation and improved phenotypic characterization of Treg cells. The obtained results pave the way for developing innovative therapeutic approaches for autoimmune diseases, graft rejection and cancer, which hold a promise to greatly improve patients´quality of life.Foxp3+ CD25+ regulatorne T stanice (Tregs) rijetka su podskupina CD4+ T stanica, koje igraju vitalnu ulogu u regulaciji imunološkog sustava. Zbog svojih imunosupresivnih sposobnosti, Treg su zanimljive kao potencijalno novo sredstvo za liječenje autoimunih bolesti i sprječavanje odbacivanja presatka. Iako je populacija Treg stanica dobro definirana unutarstaničnom ekspresijom transkripcijskog faktora Foxp3, ne postoji definirani površinski marker koji bi razlikovao Tregs od ostalih T stanica, što pouzdanu fenotipsku karakterizaciju i izolaciju čini izazovnom. Nadalje, većina protokola izolacije i ekspanzije dizajnirana je za ljudske Treg stanice, što predstavlja značajan nedostatak jer su miševi važni modelni organizmi u pretkliničkoj fazi razvoja lijeka. Primijenjen je niz metodologija. Treg stanice izolirane su iz slezena miševa pomoću imunomagnetske separacije. Stanice su zatim aktivirane i ekspandirane tijekom 15 dana, korištenjem različitih omjera Treg stanica i aktivacijskih kuglica u prisutnosti interleukin-2 citokina. Nakon bojanja antitijelima za vijabilnost, površinske i unutarstanične markere, fenotip Treg stanica i drugih podskupova T stanica analiziran je protočnom citometrijom. Kako bismo istražili transkripcijske profile subpopulacija unutar Treg uzoraka, izvršili smo 10x Genomics single-cell RNA sekvenciranje (scRNAseq). Ova studija rezultirala je uspješnom uspostavom protokola izolacije i ekspanzije za mišje Foxp3+ CD25+ Tregs, kao i definiranim sveobuhvatnim panelima kombinacije antitijela za protočnu citometriju. Analiza fenotipa i transkriptoma otkrila je heterogenost Treg populacije već pri samoj izolaciji iz sekundarnog limfnog tkiva.
ScRNA-seq analiza pružila je dublji uvid u transkriptom Treg stanica i otkrila druge važne biomarkere za izolaciju bez kontaminanata i poboljšanu fenotipsku karakterizaciju Treg stanica. Dobiveni rezultati otvaraju put prema razvoju inovativnih pristupa liječenju za autoimune bolesti, odbacivanje presatka i rak, koji obećavaju značajno poboljšanje kvalitete života pacijenata
Standardizacija i optimizacija neuralnih staničnih modela i biomarker eseja za rijetke genetičke bolesti
No full textNiemann-Pick Disease Type C (NPCD) is a rare neurodegenerative disorder affecting 1 in 120,000 live births, characterized by complex alterations in sphingolipid metabolism. While cholesterol accumulation is a prominent feature, the situation is more intricate in the brain, where primary accumulating lipids are sphingolipids such as GM2 and GM3 gangliosides. So far, the only approved therapy to date, Miglustat, targets sphingolipid pathways and is used to prevent disease progression. The objective of this study is to improve an existing ganglioside assay for its utilization in cellular models. The optimization process encompasses the careful selection of the most effective extraction buffers, optimizing the protocol steps to enhance efficiency, and selecting appropriate sample sizes for cellular pellets. This study aims to develop a universally applicable assay that can be used across a wide range of cell lines. The assay validation involves the utilization of differentiated astrocytes and neural progenitor cells derived from fibroblast lines harboring NPC1-heterozygous and NPC1-homozygous variants. Quantification of gangliosides is performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS).The findings of this investigation demonstrated a notable disparity in the buildup of GD3 ganglioside in fibroblast cell lines that were homozygous for the NPC1 gene mutation. Conversely, neural progenitor cells and astrocytes exhibited a reduction in GD3 ganglioside levels. Furthermore, it was observed that NPC1-homozygous fibroblasts exhibited increased concentrations of complex GD1a and GT1b gangliosides. Additional investigation is required to effectively address the inquiries that have been prompted by these results. This assay holds significant potential, hence redefining the application of cellular models in in vitro experiments related to lysosomal storage diseases. Upon optimization, the ganglioside assay will function as a valuable instrument for capturing the accumulation pattern of gangliosides that are linked to rare diseases.Niemann-Pickova bolest tipa C (NPCD) rijedak je neurodegenerativni poremećaj koji pogađa 1 od 120 000 živorođene djece, a karakteriziraju ga složene promjene u metabolizmu sfingolipida. Dok je nakupljanje kolesterola glavno obilježje ove bolesti, situacija je složenija u mozgu gdje se primarno nakupljaju sfingolipidi kao što su GM2 i GM3 gangliozidi. Do sada jedina odobrena terapija, Miglustat, inhibira sintezu sfingolipida i koristi se za sprječavanje progresije bolesti. Cilj rada je prenamijeniti postojeći esej gangliozida za njegovu upotrebu u staničnim modelima. Proces optimizacije obuhvaća pažljiv odabir otopine za ekstrakciju, optimiziranje koraka protokola za povećanje učinkovitosti i odabir odgovarajuće veličine uzorka za stanične pelete. Cilj ove studije je razviti univerzalno primjenjivi test koji se može koristiti u širokom rasponu staničnih linija. Validacija testa uključuje korištenje diferenciranih astrocita i neuralnih progenitorskih stanica diferenciranih iz linija fibroblasta koje sadrže NPC1-heterozigotne i NPC1-homozigotne varijante. Kvantifikacija gangliozida provodi se pomoću tekućinske kromatografije-tandem masene spektrometrije (LC-MS/MS). Rezultati ovog istraživanja pokazali su primjetnu razliku u nakupljanju GD3 gangliozida u linijama fibroblasta koje su bile homozigotne za mutaciju gena NPC1. Suprotno tome, neuralne progenitorske stanice i astrociti pokazali su smanjenje razine GD3 gangliozida. Nadalje, primijećeno je da NPC1-homozigotni fibroblasti pokazuju povećane koncentracije kompleksnih GD1a i GT1b gangliozida. Dodatna istraživanja su potrebna kako bi se učinkovito odgovorilo na upite koje su potaknute ovim rezultatima. Ovaj test ima značajan potencijal, stoga redefinira primjenu staničnih modela u in vitro eksperimentima koji se odnose na lizosomske bolesti nakupljanja. Nakon optimizacije, analiza gangliozida funkcionirat će kao vrijedan instrument za mjerenje gangliozida koji su povezani s rijetkim bolestima
Role of interferon gamma in the control of mouse cytomegalovirus infection in neurons
No full textCytomegalovirus (CMV) is a highly prevalent β-herpesvirus that infects the majority of the population worldwide. CMV infections are usually asymptomatic, however, in immunocompromised individuals and newborns they can lead to severe sequelae and life-threatening conditions. Congenital CMV infection is the most common congenital infection that can affect brain development and cause severe neurological disabilities. CMV can infect a broad range of cells including neurons, which employ pro-survival strategies to avoid destruction from the immune system. Generally, CD4+ and CD8+ T cells play a crucial role in the control of CMV infection in the brain. Since these cells secrete IFNγ, the objective of this thesis is to determine the role of IFNγ in the control of MCMV infection in neurons, using a mouse model of congenital infection. For this purpose, newborn mice lacking IFNγR in neurons were infected with MCMV and organs were harvested at three timepoints of infection. Viral titers in brain were determined by plaque assay and the percentage of infected neurons was quantified by dual immunohistochemical analysis. Analysis of viral titers was also performed for congenitally infected mice lacking IFNγR in astrocytes. The results of these experiments show that IFNγ does not directly control MCMV infection in neurons and astrocytes, both during, and after resolution of productive infection. These findings add to the understanding of immune responses to CMV infection which is pivotal for development of effective therapies and vaccines and raise new questions on which molecules and cells are involved in the control of CMV infection in neurons.Citomegalovirus (CMV) je široko rasprostranjen β-herpesvirus koji inficira većinu populacije širom svijeta. CMV infekcije su obično asimptomatske, međutim u imunokompromitiranih pojedinaca i novorođenčadi mogu dovesti do ozbiljnih posljedica te stanja opasnih po život. Kongenitalna CMV infekcija je najčešća kongenitalna infekcija koja može utjecati na razvoj mozga i uzrokovati ozbiljne neurološke poremećaje. CMV može inficirati širok raspon stanica, uključujući neurone koji koriste strategije preživljavanja kako bi izbjegli uništenje od strane imunološkog sustava. Općenito, CD4+ i CD8+ T stanice imaju presudnu ulogu u kontroli CMV infekcije u mozgu. Budući da te stanice izlučuju IFNγ, cilj ovog rada je utvrditi ulogu IFNγ u kontroli MCMV infekcije u neuronima, koristeći mišji model kongenitalne infekcije. U tu svrhu, novorođeni miševi kojima nedostaje IFNγR u neuronima inficirani su MCMV-om te su im organi prikupljeni u tri vremenske točke infekcije. Virusni titar u mozgu je određen analizom plakova te je kvantificiran postotak inficiranih neurona analizom dvojne imunohistokemije. Analiza virusnih titrova provedena je i za kongenitalno-inficirane miševe kojima nedostaje IFNγR u astrocitima. Rezultati ovih eksperimenata pokazuju da IFNγ nema direktnu kontrolu nad MCMV infekcijom u neuronima i astrocitima tijekom te nakon rezolucije produktivne infekcije. Ovi pronalasci pridodaju razumijevanju imunoloških odgovora na CMV infekciju, što je ključno za razvoj učinkovitih terapija i cjepiva te postavljaju nova pitanja o tome koje molekule i stanice su uključene u kontrolu CMV infekcije u neuronima
The effect of PI4KIIa and PI4KIIIb on platelet cytoskeleton
No full textTrombociti su krvne stanice koje nastaju oslobađanjem dijelova citoplazme zrelih megakariocita. U cirkulaciji se nalaze u stanju mirovanja, međutim pronalaskom oštećenja krvne žile, oni se aktiviraju što vodi promjeni oblika te stvaranju ugruška. Uslijed stimulacije trombocita dolazi do polimerizacije aktina, a adhezija trombocita ostvaruje se stvaranjem adhezivnih produžetaka: filopodija i lamelipodija. Procesi remodeliranja membrane i vezikularni promet potrebni su za aktivaciju trombocita te su posredovani i fosfoinozitidima (PI), fosforiliranim membranskim lipidima. Najobliniji PI u stanici su fosfatidilinozitol-4,5-bifosfat (PI(4,5)P2) i fosfatidilinozitol-4-monofosfat (PI4P), te se smatra da je PI4P glavni izvor PI(4,5)P2. PI4P mogu proizvoditi fosfatidilinozitol 4- kinaze tipa II (PI4KIIα i PI4KIIβ) i kinaze tipa III (PI4KIIIα i PI4KIIIβ). U ovom radu, pokazujemo kako specifična farmakološka inhibicija PI4KIIIβ, IN-9 inhibitorom, smanjuje širenje trombocita na kolagenu. Inhibirani trombociti su okruglastiji i nerašireni, pokazuju nemogućnost polimerizacije F-aktina uočeno po povećanom broju filopodija u odnosu na lamelipodije te izostanak aktinomiozinskih stresnih vlakana koja pomažu u održavanju širenja trombocita. Nadalje, imunofluorescencijom smo pokazali lokalizaciju PI4KIIα u mirujućim i aktiviranim trombocitima i njenu kolokalizaciju s α-tubulinom. Također, pokazujemo kako inhibicija PI4KIIα inhibitorom PI-273, tek u najvećoj koncentraciji djelomično smanjuje širenje trombocita na kolagenu ukazujući na slabiju ulogu ove kinaze u ovom aspektu funkcije trombocita.Platelets (PLT) are blood cells that are formed by the release of parts of the cytoplasm of mature megakaryocytes. In the circulation, they are in their resting state, but upon vessel wall injury, they activate, which leads to PLT shape change and the formation of a thrombus. Actin polymerization occurs as a result of PLT activation, and PLT adhesion is achieved by the formation of adhesive extensions: filopodia and lamellipodia. Membrane remodeling and vesicular trafficking are needed for PLT activation, and these processes are mediated also by phosphoinositides (PIs), phosphorylated membrane lipids. Two most abundant PIs in the cells are phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol-4-monophosphate (PI4P), the later being a major source for PI(4,5)P2. PI4P can be produced by type II phosphatidylinositol 4-kinases (PI4KIIα and PI4KIIβ) and type III kinases (PI4KIIIα and PI4KIIIβ). In this study, we show that specific pharmacological inhibition of PI4KIIIβ, with inhibitor IN-9, reduces PLT spreading on collagen. Inhibited platelets are more rounded and unexpanded, exhibit an inability to polymerize F-actin as evidenced by an increased number of filopodia compared to lamellipodia, and the absence of actinomyosin stress fibers that help maintain spreaded platelet. Furthermore, we showed the localization of PI4KIIα in resting and activated platelets and its colocalization with α-tubulin using immunofluorescence. We also show that inhibition of PI4KIIα with PI-273 mildly only at higher concentrations reduces platelet spreading on collagen. indicating a weaker role of PI4KIIα in this aspect of platelet function
Preparation of meso-(N, N-diethylaminophenyl)porphyrins and their N-oxidation
No full textFotodinamička terapija (PDT) je inovativni tretman koji selektivno cilja maligne stanice uz minimalnu štetu zdravom tkivu, čineći je učinkovitom za tumore i određene kožne bolesti. Postupak uključuje akumulaciju fotosenzibilizatora (PS) u tumorskom tkivu, osvjetljavanje zahvaćenog područja određenom valnom duljinom što rezultira stvaranjem reaktivnih kisikovih spojeva (ROS). Iako općenito neučinkovita protiv metastaza, PDT se može kombinirati s drugim terapijama kao što su kemoterapija ili radioterapija kako bi se poboljšala njezina učinkovitost i prevladali njeni nedostaci. Za učinkovitu terapiju ključno je odabrati optimalan fotosenzibilizator koji je dobro topiv u vodenim otopinama, netoksičan u mraku, učinkovito apsorbira u vidljivom dijelu spektra te učinkovito proizvodi singletni kisik i drugih ROS. Vrlo je bitan optimalan omjer lipofilnih i hidrofilnih dijelova u strukturi fotosenzibilizatora kako bi se olakšala akumulacija u tumorskim stanicama, što se postiže modifikacijama početnih struktura fotosenzibilizatora.
Simetrični meso-tetra-(4-dietilaminofenil)porfirin i njegovi asimetrični derivati u kombinaciji s 4-acetamidofenilnim supstituentima uspješno su izolirani za upotrebu kao fotosenzibilizatori u PDT-u. Sve nove strukture i njihova čistoća potvrđeni su 1H i 13C NMR spektroskopijom. Spojevi su pokazali apsorpciju u crvenom dijelu spektra, izuzetnu fotostabilnost i učinkovitu proizvodnju singletnog kisika, osobito 5-[4-(N-acetamido)fenil]-10,15,20-tri(4-(N,N-dietilamino)fenil)porfirin(A3B). Fototoksičnost sintetiziranih PS-ova ispitivana je MTT testom uz osvjetljavanje crvenim svjetlom (λ = 643 nm, 2 mW/cm2) na dvije tumorske stanične linije, MDA-MB 231 i HeLa, te na HFF kao normalnoj staničnoj liniji. Simetrični 5,10,15,20-tetra(N,N-dietil-4-aminofenil)porfirin (A4) zbog slabe topljivosti nije pokazao citotoksičan učinak, dok A3B, unatoč najefikasnijoj generaciji singletnog kisika, pokazao je smanjenu citotoksičnost u usporedbi sa 5,15-di[4-(N-acetamido)fenil]-10,20-di(4-(N,N-dietilamino)fenil)porfirin (trans-A2B2) i 5,10-di[4-(N-acetamido) fenil]-15,20-di(4-(N,N-dietilamino)fenil)porfirin (cis-A2B2) koji su pokazali najjaču citotoksičnost, ali ne i selektivnost prema tumorskim stanicama. Potrebne su daljnje modifikacije struktura dobivenih porfirina kako bi se poboljšala svojstva i postigli učinkovitiji fotosenzibilizatori.Summary
Photodynamic therapy (PDT) is an innovative treatment that selectively targets malignant cells while causing as little damage as possible to healthy tissue and it is, therefore, effective for tumors and certain skin diseases. The process involves the accumulation of photosensitizer (PS) in the tumor tissue, the illumination of the affected area with a specific wavelength and the generation of reactive oxygen species (ROS). Although PDT is generally ineffective against metastases, it can be combined with other therapies such as chemotherapy or radiotherapy to increase its effectiveness and overcome its limitations. For effective therapy, it is crucial to select an optimal PS that is highly soluble in aqueous solutions, non-toxic in the dark, has effective absorption in the visible spectrum, and efficiently generates singlet oxygen and ROS. The optimal ratio of lipophilic and hydrophilic parts in the structure of the PS is crucial for accumulation in tumor cells, which is achieved by various modifications of the original PS structure.
Symmetric meso-tetra-(4-diethylaminophenyl)porphyrin and its asymmetric derivatives with 4-acetamidophenyl substituents were successfully isolated for use as PSs in PDT. All new structures and their purity were confirmed by 1H and 13C NMR spectroscopy. They showed absorption in the red region of the spectrum, exceptional photostability and effective singlet oxygen production, especially the 5-[4-(N-acetamido)phenyl]-10,15,20-tri(4-(N,N-diethylamino) phenyl)porphyrin (A3B). The phototoxicity of the PSs was investigated using red light and MTT assay (λ = 643 nm, 2 mW/cm2) on two tumor cell lines, MDA-MB 231 and HeLa, and HFF as a normal cell line. Symmetric 5,10,15,20-tetra(N,N-diethyl-4-aminophenyl)porphyrin (A4) showed no cytotoxic effect due to its poor solubility, while A3B showed no cytotoxic effect despite its highly efficient production of singlet oxygen, in contrast to 5,15-di[4-(N-acetamido)phenyl]-10,20-di(4-(N,N-diethylamino)phenyl)porphyrin (trans-A2B2) and 5,10-di[4-(N-acetamido)phenyl]-15,20-di(4-(N,N-diethylamino) phenyl)porphyrin (cis-A2B2)which showed cytotoxic effects but no selectivity towards tumor cells. Further modifications of the obtained porphyrin structures are required to improve their properties and to develop more effective photosensitizers
Razvoj novih protokola za izolaciju polietilen tereftalata i polistirena iz morskih uzoraka te njihova identifikacija pomoću ATR-FTIR-a i MALDI-MS-a
No full textPolymers are macromolecules with repeated structural units. Synthetic polymers have first been introduced in the middle of the 19th century, and since then their production has exponentially increased on yearly basis. As of 2015, more than 6300 Mt of plastic waste has been generated throughout the world, with more than 76% of that waste ending in landfills and oceans. To fully estimate the effect of this pollution on our planet, simple standardized protocols should be set in place for microplastic analysis. This study presents a novel approach for isolating polyethylene terephthalate (PET) and polystyrene (PS) particles and their quantification and identification using Fourier transformation infrared spectroscopy (FTIR) and matrix-assisted laser desorption ionization (MALDI) mass spectrometry. Additionally, seawater samples were analyzed to determine and optimize the microplastic isolation protocol using chemical digestion of the organic matter coupled with density separation of microplastic from sodium metatungstate (SPT) saline solution. Chemical digestion of the organic matter from seawater samples was tested with both hydrogen chloride (HCl) and hydrogen peroxide (H2O2) solutions. FTIR was later used to determine minimal concentration of PET and PS in seawater needed for their identification. Acquired minimal concentration was further tested with reagents used in chemical digestion to assess the effect of various solutions on PET and PS particles identification. The MALDI method for PET and PS identification was used to study the effect of different polymer suspension concentrations and two different cationization agents, sodium iodide (NaI) and silver trifluoroacetate (AgTFA). Identification of both PS and PET with FTIR in different solutions as well as the development of the MALDI method for PET identification were successful.Polimeri su makromolekule koje sadrže ponavljajuće strukturne jedinice. Sintetički polimeri su se prvi puta pojavili sredinom 19. stoljeća te od tada njihova proizvodnja eksponencijalno raste na godišnjoj razini. Od 2015. godine, više od 6300 Mt plastičnog otpada je nastalo diljem svijeta, s više od 76% od toga otpada odbačenog na odlagališta smeća i u mora. Kako bi u potpunosti mogli procijeniti učinak ovog zagađenja na naš okoliš i nas same, jednostavni standardizirani protokoli bi trebali biti razvijeni za analiziranje čestica mikroplastike. Ovaj rad prikazuje novi postupak u izoliranju čestica polietilen tereftalata (PET) i polistirena (PS) i njihovu identifikaciju pomoću Fourierove transformirane infracrvene spektroskopije (FTIR) i matricom asistirane laserske desorpcije/ionizacije (MALDI) masene spektrometrije. Također, morski uzorci su analizirani kako bi odredili i optimizirali izolacijski protokol koji koristi kemijsku digestiju organskih tvari i razdvajanje mikroplastike temelju gustoće iz slane otopine natrijevog polivolframata (SPT). Kemijska digestija organskih tvari iz uzoraka morske vode je testirana sa klorovodičnom kiselinom (HCl) i vodikovim peroksidom (H2O2). FTIR je potom korišten da bi se odredila minimalna koncentracija PET-a i PS-a u morskoj vodi potrebna za njihovu identifikaciju. Dobivena minimalna koncentracija je potom testirana sa reagensima korištenima u kemijskoj digestiji da bi se provjerio učinak različitih otopina na identifikaciju čestica PET-a i PS-a. Korištena MALDI metoda za identifikacija PET-s i PS-a usporedila je utjecaj različitih koncentracija polimera u otopini i dvaju različitih kationizacijskih agensa, natrijevog jodida (NaI) i srebrovog trifluoroacetata (AgTFA), na rezultate analize. Identifikacija PS-a i PET-a sa FTIR-om u različitim otopinama i razvoj MALDI metode za identifikaciju PET-a su bili uspješni
Isolation, characterization and evaluation of biological activities of extracts and natural compounds isolated from the Adriatic sea coral, Eunicella cavolini
No full textMorski sjedilački organizmi poput spužvi i koralja prilagodili su se ekstremnim uvjetima morskog okoliša sintezom sekundarnih metabolita s jedinstvenim strukturnim karakteristikama koji su pokazali široki spektar bioloških aktivnosti in vitro i in vivo. U prijašnjim studijama iz oktokoralja roda Eunicella, izolirani su spojevi iz klase steroida, terpenoida i alkaloida s prvenstveno citotoksičnim učincima. Unatoč tome što su Eunicella koralji iz Sredozemnog mora bili predmet nekolicine istraživanja unazad nekoliko desetljeća, oni iz Jadranskog mora do sada nisu istraživani u cilju izolacije prirodnih spojeva i biološke evaluacije istih. Stoga, je ovim doktorskim istraživanjem po prvi puta provedena kemijska karakterizacija metabolita i biološka evaluacija ekstrakta te polu-pročišćenih frakcija dobivenih iz koralja Eunicella cavolini (Koch, 1887.), s ciljem utvrđivanja potencijala antiproliferativnih, antioksidativnih, antimikrobnih i protuupalnih učinaka. Preliminarni podaci su pokazali kako ekstrakti i frakcije koralja pokazuju umjereni antiproliferativni učinak na panelu tumorskih stanica i djelovanje prema Gram-pozitivnim bakterijama, kao i toksične učinke u razvoju embrija zebrica Danio rerio. Stoga, je iz frakcije F1 izoliran i okarakteriziran steroid pregnanskog tipa kojemu su po prvi puta ispitane biološke aktivnosti te je dodatno pripremljen derivat modifikacijom supstituenta. Ti su spojevi pokazali potencijalno protuupalno djelovanje inhibicijom lipooksidaze iz soje te denaturacije albumina uz umjerenu inhibiciju rasta tumorskih stanica i selektivnost prema stanicama adenokarcinoma debelog crijeva. Također je prirodni pregnanski produkt imao zapaženo antibakterijsko djelovanje (MIC = 62,5 µM na B. subtilis) dok su i pregnan i njegov deriviat pokazali toksični efekt na razvoj zebrica pri niskim koncentracijama. S druge strane, doktorski rad je uključivao pripravu jednog od metabolita koji se prirodno pojavljuje u koralju roda Eunicella, granulatamida B te je priređeno desetak njegovih strukturnih analoga modulirajući pobočni masnokiselinski lanac (duljinu lanca, zasićenost, konjugaciju dvostrukih veza). Pokazalo se da granulatamid B i analozi s nezasićenim i/ili konjugiranim dvostrukim vezama uzrokuju umjerenu inhibiciju rasta tumorskih stanica, pri čemu je analog sa lancem retinoične kiseline pokazao antioksidativno djelovanje usporedivo s pozitivnom kontrolom. Dodatno, samo je granulatamid B imao antibakterijsko djelovanje i to prema Gram-pozitivnom B. subtilis. Obzirom na to da strukturni analog granulatamida B sa α-linolenskim lancem nije uzrokovao abnormalan razvoj zebrica, te je imao selektivnije djelovanje prema tumorskim stanicama, mogao bi predstavljati molekulu od interesa za daljnju promjenu strukture i postizanje boljih bioloških učinaka. U konačnici, ovim se istraživanjem otvara put daljnjim ispitivanjima biološkog potencijala ekstrakta, odnosno spojeva jadranskog koralja, E. cavolini s naglaskom na kemijsku modifikaciju struktura s ciljem smanjenja toksičnosti uzoraka.Sedentary marine organisms such as sponges and corals have adapted to the extreme conditions of the marine environment by synthesizing secondary metabolites with unique structural characteristics that have shown a wide range of biological activities in vitro and in vivo. Furthermore, compounds from the class of steroids, terpenoids and alkaloids with primarily cytotoxic effects were isolated via bioprospecting programmes from octocorals of the genus Eunicella. Even though Eunicella corals from the Mediterranean Sea have been the subject of several studies over the past few decades, those from the Adriatic Sea have not been investigated so far with the aim of isolating natural compounds and their biological evaluation. Therefore, within this doctoral research, chemical characterization of metabolites and biological evaluation of extracts and semi-purified fractions obtained from the coral Eunicella cavolini (Koch, 1887) were carried out for the first time, with the aim of determining the potential of antiproliferative, antioxidant, antimicrobial and anti-inflammatory effects. Preliminary data showed that coral extracts and fractions show a moderate antiproliferative effect on a panel of tumour cells and activity against Gram-positive bacteria. Also, toxic effects were observed in the development of zebrafish (Danio rerio) embryos. Consequently, a pregnane-type steroid was isolated and characterized from fraction F1, whose biological activities were tested for the first time, and a derivative was additionally prepared by modifying the substituent. These compounds showed potential anti-inflammatory activity by inhibiting soy lipoxygenase and albumin denaturation with moderate inhibition of cancer cells growth and selectivity towards colon adenocarcinoma cells. Natural pregnane had a noticeable antibacterial effect (MIC = 62.5 µM on B. subtilis) while both pregnane and its derivative exerted toxic effects on zebrafish development were also observed at low concentrations. On the other hand, the doctoral thesis included the preparation of one of the metabolites that occurs naturally in the coral genus Eunicella, granulatamide B, and a dozen of its structural analogues were prepared by modulating the fatty acid side chain (chain length, saturation, conjugation of double bonds). Granulatamide B and analogues with unsaturated and/or conjugated double bonds were shown to cause moderate inhibition of cancer cells growth, with the analogue with a retinoic acid chain showing antioxidant activity comparable to the control. Additionally, only granulatamide B had antibacterial activity against Gram-positive B. subtilis. Given that the structural analogue of granulatamide B with an α-linolenic chain did not cause abnormal development of zebrafish and had a more selective effect on cancer cell lines, it could represent a molecule of interest for further structural change in order to achieve better biological effects. Finally, this research paves the way for further investigation of the biological potential of the extract, or compounds of the Adriatic coral, E. cavolini with an emphasis on the chemical modification of the structures with the aim of reducing the toxicity of the samples