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Analysis of NK cells in CD16 deficient animals
No full textPrirodnoubilačke ili stanice NK (engl. natural killer cells, NK cells) važna su sastavnica urođenog imunološkog sustava zbog svoje sposobnosti brzog prepoznavanja i eliminacije tumorskih i virusom zaraženih stanica. Njihova aktivacija ovisi o ravnoteži signala primljenih preko aktivacijskih i inhibicijskih receptora. Jedan od aktivacijskih receptora izraženih na stanicama NK je i CD16 receptor, poznat po svojoj ulozi u staničnoj citotoksičnosti posredovanoj protutijelima (engl. antibody-dependent cellular cytotoxicity, ADCC). Međutim, uloga ovog receptora u drugim aspektima biologije stanica NK još uvijek nije dovoljno istražena. Stoga je naš glavni cilj bio ispitati utjecaj nedostatka izražaja CD16 receptora na stanice NK u različitim organima. Koristili smo genetski modificirane miševe kojima nedostaje izražaj CD16 receptora i usporedili ih s miševima divljeg soja. Proveli smo fenotipsku i funkcionalnu analizu stanica NK. Uočili smo da nedostatak izražaja CD16 receptora dovodi do promjene u sazrijevanju stanica NK, kao i do promjena u njihovom postotku i broju u koštanoj srži. Nedostatak izražaja ovog receptora nije doveo do promjene u izražaju većine analiziranih aktivacijskih i inhibicijskih receptora, izuzev NCR1 receptora čiji je izražaj smanjen kod CD16 deficitnih životinja. Također, CD16 deficitne stanice NK nisu pokazale razliku u sposobnosti produkcije citokina u odnosu na kontrolnu grupu. Rezultati ovoga rada ukazuju na dosad nepoznatu ulogu CD16 receptora u sazrijevanju stanica NK iako su potrebna dodatna istraživanja kako bi se ta uloga do kraja istražila.Natural killer (NK) cells are an important component of the innate immune system due to their ability to rapidly recognize and eliminate tumor cells and virus-infected cells. Their activation depends on a balance of signals received through activating and inhibitory receptors. One key activating receptor expressed on NK cells is the CD16 receptor, known for its role in antibody-dependent cellular cytotoxicity (ADCC). However, the role of this receptor in other aspects of NK cell biology remains largely unexplored. The main aim of this study was to investigate the impact of CD16 receptor deficiency on NK cells in different organs. We used genetically modified mice lacking CD16 expression and compared them to wild-type mice. We performed phenotypic and functional analyses of NK cells. We observed that the absence of CD16 expression leads to changes in NK cell maturation, as well as changes in their percentage and number in the bone marrow. The absence of this receptor did not result in changes in the expression of most analysed activating and inhibitory receptors, except for the NCR1 receptor, which showed reduced expression in CD16-deficient animals. Additionally, CD16-deficient NK cells did not show differences in cytokine production capacity compared to the control group. These findings indicate a previously unknown role of the CD16 receptor in NK cell maturation, although further studies are needed to fully elucidate this role
Contribution of different estrogen receptor alpha (ER) domains in ER-driven transcriptomic profile of mammary epithelial cells and characterization of the ER-PR proximity in ER+ breast cancer
No full textAim of the study: 70% of breast cancers (BC) are estrogen receptor alphapositive (ER+), and typically treated by targeting ER signaling. Previous research showed that membrane ER, as well as activation function 1 (AF-1) and activation function 2 (AF-2) domains are involved in the mammary gland and BC development. We aimed to characterize the contribution of each ER domain in ER regulated transcriptional profile in mammary epithelial cells (MEC). First, we researched whether ER-dependent gene
expression is domain-specific. We tested the hypothesis that pathways relevant for BC are ER domain-dependent, and aimed to characterize whether those pathways are regulated by the cooperation of all domains or by an individual domain. We focused on the dependences of the expression of ER target genes, such as Areg, Greb1, and Pgr on different ER domains. Finally, we aimed to characterize the levels of interactions between PR and ER in cancer cell lines MCF7 and T47D. Material and methods: In this
thesis, RNAseq data of GFP+ MECs from C57Bl6 mice lacking complete ER, membrane ER, AF-1 or AF-2 ER domains were analyzed in R software. MCF7 and T47D cell lines were treated with 100 nM progesterone (P4) to establish the conditions suitable for ER-PR interactions. The expression of Pgr was measured by qPCR and ER-PR interactions were analyzed by proximity ligation assay. Results and conclusions: Each ER domain strongly contributes to ER-regulated gene expression in MECs. To regulate pathways involved in carcinogenesis, cell proliferation, and immune response, ER domains act synergically, but also, in some pathways, an individual domain plays a regulatory role of the whole receptor. ER-PR direct interactions occur
in nuclei, but also in non-nuclear compartments in MCF7 and T47D cells with an increased number of interactions upon P4 treatment
Pokret u integriranom kurikulumu - povezanost motoričkih i istraživačkih aktivnosti kod djece rane dobi
No full textKod djece rane dobi sve je prisutniji nedostatak osnovne motoričke pismenosti koja je iznimno bitna u istraživačkim aktivnostima djece te je izravno povezana s razvojem centralnog živčanog sustava. Dva su cilja istraživanja. Prvi je utvrditi razlike između odgojne skupine u kojoj se provodi integrirano učenje djece i odgojne skupine u kojoj se odgojno-obrazovni rad temelji na integriranom učenju uz pokret. Drugi cilj je utvrditi utjecaj različitih pristupa integriranom učenju djece na njihovu razinu razvoja motoričkih postignuća. Istraživanjem su obuhvaćene dvije mješovite jasličke skupine djece iz dječjih vrtića Grada Rijeke, u eksperimentalnoj skupini 18 djece (Vrtić A) i u kontrolnoj skupini 16 djece (Vrtić B). Kvalitativne varijable kodirane su na temelju značajki integriranog učenja i dimenzija suvremenog kurikuluma (prilagođeno prema Petrović-Sočo, 2009) odnosno aktivnosti koje su organizirane u prostorima: soba dnevnog boravka, hodnik, dvorana i otvoreni (vanjski) prostor. Aktivnosti analiziramo sastavnicama: organizacija prostora, odgajatelj i primjena odgojno-obrazovnih strategija, slika o djetetu, sredstva, materijali i igračke i ozračje u odgojnoj skupini. Kvantitativne varijable formirane su na temelju četiriju testova za mjerenje motoričkih postignuća djece rane i predškolske dobi prema Petrić i sur. (2018). Rezultati ovoga istraživanja ukazuju na značajne razlike između djece odgojnih skupina u kojima se provodi integrirano učenje odnosno integrirano učenje uz pokret. Obje skupine, eksperimentalna i kontrolna, odgojno-obrazovni proces temelje na integriranom pristupu u planiranju odgojno-obrazovnog rada, ali u eksperimentalnoj skupini naglasak je na aktivnostima djece koje su povezane s pokretom.With young children, there is a growing lack of basic motor literacy, which is extremely important in the research activities of children and is directly related to the development of the central nervous system. There are two research goals. The first is to identify the differences between the educational group in which integrated children's learning is conducted and the educational group in which educational work is based on integrated learning with movement. The second objective is to determine the impact of different approaches to integrated learning on their level of motor achievement. The study included two mixed nursery groups from kindergartens in the city of Rijeka, in the experimental group of 18 children (Kindergarten A) and in the control group of 16 children (Kindergarten B). Qualitative variables are coded on the basis of integrated learning features and dimensions of the contemporary curriculum (adapted according to Petrović-Sočo, 2009) and activities organized in the spaces: living room, hall, gym and open (outdoor) space. We analyze the activities with the following components: organization of space, educator and application of educational strategies, image of the child, resources, materials and toys and the atmosphere in the educational group. Quantitative variables were formed based on four tests for measuring motor achievement of young children and preschoolers according to Petrić et al. (2018). The results of this study indicate significant differences between the children from the educational groups in which integrated learning and integrated learning with movement are conducted. Both groups, experimental and control, base the educational process on an integrated approach in planning educational work, but in the experimental group the emphasis is on the activities of children associated with movement
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
Redesign of the Impactor for Total Hip Arthroplasty
No full textThis master's thesis demonstrates the proper approach to problem-solving by carefully evaluating all the details and establishing guidelines in collaboration with surgeons to create the best product on the market. The foundation of this project was laid by compiling a list of functions, requirements, and desires with the help of expert reports and surgeon feedback after brainstorming, sketching different ideas, and exploring various options to improve the current impactor or develop a new solution. However, it was determined that the current version would require too much work to optimize and would not fulfill the demands as some other designs might. Therefore, a new idea was developed by observing competitors and creating a similar but different solution compatible with the shape of EcoFit 2M implants. After numerous prototypes and iterations, the redesign addressed all the functions, requirements, and wishes set at the beginning of this task and objectively made an excellent instrument, the impactor for total hip arthroplasty, hopefully improving the quality and outcomes of orthopedic surgeries
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
Pokret u integriranom kurikulumu – utjecaj prostornog uređenja na razinu kretanja u istraživačkim aktivnostima djece rane dobi
No full textSuvremena istraživanja sve veći naglasak stavljaju na važnost oblikovanja učinkovitih i inovativnih okruženja za integrirano učenje ističući kako prostor u kojemu djeca borave treba učiniti što funkcionalnijim i višenamjenskim. Ovo istraživanje ima dva cilja. Prvi, utvrditi razlike između suvremenog prostornog okruženja i suvremenog prostornog okruženja koje potiče pokret. Drugi, utvrditi razlike u razini tjelesne aktivnosti između djece u kontrolnoj skupini koji borave u suvremenom prostornom okruženju i djece u eksperimentalnoj skupini koji borave u suvremenom prostornom okruženju koje potiče pokret. U istraživanju su sudjelovala dva novoizgrađena i dječja vrtića sa suvremenom arhitekturom vanjskog i nutarnjeg prostora u Gradu Rijeka. U svakom od navedenih dječjih vrtića istraživanjem je obuhvaćena jedna mješovita jaslička skupina. Kvalitativne varijable kodirane su na temelju prostora u kojima djeca svakodnevno borave, i to: soba dnevnog boravka, hodnik, dvorana i otvoreni (vanjski) prostor. Kvantitativne varijable formirane su na temelju mjernog instrumenta pedometra Omron HJ-720IT-E2. Rezultati su ukazali na značajnu razliku (p=0,00) u korist suvremenog prostornog okruženja koje potiče pokret. Djeca iz eksperimentalne skupine su statistički značajno tjelesno aktivniji te se u prosjeku kreću do pet puta više u odnosu na djecu iz kontrolne skupine i to na razini intenziteta koji osigurava značajni zdravstveni učinak. Ovako organizirano suvremeno prostorno okruženje koje potiče pokret, primjereno je svakom djetetu i njegovim potencijalima jer djecu ravnomjerno homogenizira u razini tjelesne aktivnosti te gotovo da ne postoje razlike među djecom eksperimentalne skupine u rezultatima razine tjelesne aktivnosti. Ovo istraživanje je iznimno bitno za neposredan rad odgajatelja i odgojno – obrazovni proces. Postavljeni su temelji iz kojih se mogu razvijati kriteriji za novi pristup organizacije prostora i integriranog učenja koje potiče pokret. Radi se o suvremenom pristupu koji dokazano više motivira dijete na tjelesnu aktivnost, omogućuje njegov cjeloviti razvoj, integrirano učenje, primjenjiv je u svim prostorima ustanove ranog odgoja i ima značajan zdravstveni učinak.Modern research places increasing emphasis on the importance of designing effective and innovative environments for integrated learning, emphasizing that the space in which children spend their time should be made as functional and multifaceted as possible. This research had two aims. The first was to identify the differences between the contemporary spatial environment and the contemporary spatial environment stimulated by movement. Secondly, it aimed at identifying differences in the level of physical activity between children in the control group who spent their time in the contemporary spatial environment and children in the experimental group who did the same, but in the contemporary spatial environment stimulated by movement. Two newly built kindergartens with contemporary outdoor and indoor facilities in the City of Rijeka participated in the study. In each of these kindergartens, a mixed nursery group was included in the study. Qualitative variables were coded based on the spaces in which children resided daily, namely the living room, hall, hall and open (outdoor) space. Quantitative variables were formed based on the Omron HJ-720IT-E2 pedometer. The results indicate a significant difference (p = 0.00) in favor of the contemporary spatial environment that supports movement. The children in the experimental group are statistically significantly more physically active, moving on average up to five times more than the children in the control group, at an intensity level that ensures a significant health effect. Such a modern spatial environment that stimulates movement is appropriate for every child and his or her potential because it homogenizes children equally at the level of physical activity and there are almost no differences between the children of the experimental group in the results of physical activity levels. This research is extremely important for the direct work of educators and the educational process. The foundations have been laid out from which criteria can be developed for a new approach to space organization and movement-driven integrated learning. It is a contemporary approach that is proven to give more motivation to children’s physical activity, thus enabling their full development and integrated learning. It is applicable in all areas of an early childhood institution and has a significant health effect
Functional Hydrogels Based on Fmoc-Amino Acids
No full textHidrogelatori male molekulske mase temeljeni na Fmoc-amino kiselinama predstavljaju obećavajuće molekule, nudeći brojne prednosti poput jednostavne pripreme hidrogelova, prilagodljivih svojstava i biokompatibilnosti. Ovo istraživanje usmjereno je na dizajn i karakterizaciju hidrogelova temeljenih na Fmoc-histidinu i Fmoc-cisteinu, kao gradivnim jedinicama koje su sposobne formirati supramolekularne strukture putem nekovalentnih interakcija kao što su vodikove veze i π-π slaganje. Histidin, polarna aminokiselina, često se koristi za katalizu u kombinaciji s cisteinom, neutralnom aminokiselinom poznatom po svojoj sklonosti stvaranju disulfidnih mostova oksidacijom. U ovoj studiji, hidrogelovi su dobiveni pri koncentracijama od 10 mM i 15 mM Fmoc-Cys u PBS-u, a kristali su dobiveni pri 10 mM Fmoc-His na temelju samoorganizacije pojedinačnih Fmoc-amino kiselina. Nadalje, njihova ko-samoorganizacija pri koncentracijama od 20 mM i 30 mM rezultirala je prozirnim hidrogelovima. Procijenjena je stabilnost hidrogelova, strukturna svojstva i katalitička aktivnost. Fmoc-His se samoorganizirao u kristale, a njihova struktura je riješena korištenjem rendgenske difrakcije. Stabilnost gela Fmoc-Cys i Fmoc-Cys:Fmoc-His ispitana je pomoću testa inverzije bočice, termoreverzibilnog testa i reologije. Osim toga, provedene su LC-MS analiza i Ellmanov test kako bi se istražila oksidacija Fmoc-Cys, dok je fluorescencijska analiza otkrila kritične koncentracije agregacije (CAC) Fmoc-amino kiselina. p-NPA test korišten je za procjenu katalitičke aktivnosti hidrogelova. Rezultati sugeriraju da ko-samoorganizirani hidrogeli Fmoc-Cys:Fmoc-His pokazuju superiornu katalitičku aktivnost u usporedbi sa samoorganiziranim Fmoc-Cys. Niža aktivnost Fmoc-Cys može se pripisati formiranju disulfidnih mostova putem oksidacijskih procesa, što smanjuje njegovu nukleofilnu reaktivnost. Ova studija doprinosi razumijevanju hidrogelatora male molekulska mase i njihovih potencijalnih primjena kao minimalističkih katalizatora.Low molecular weight hydrogelators based on Fmoc-amino acids are promising molecules, offering numerous advantages such as ease of hydrogel preparation, tunable properties and biocompatibility. This research focuses on the design and characterization of hydrogels based on Fmoc-Histidine and Fmoc-Cysteine, as building blocks capable of forming structures supramolecular through non-covalent interactions such as hydrogen bonding and π-π stacking. Histidine, a polar amino acid, is frequently exploited for catalysis in combination with cysteine, a neutral amino acid known for its propensity to create disulfide bridges through oxidation. In this study, hydrogels are obtained at 10 mM and 15 mM of Fmoc-Cys in PBS and crystals are obtained at 10 mM of Fmoc-His based on the self-assembly of individual Fmoc-amino acids. Furthermore, their co-assembly at 20 mM and 30 mM resulted in transparent hydrogels. Hydrogel stability, structural properties and catalytic activity were assessed. Fmoc-His self-assembled into crystals and its structure was solved using X-ray diffraction. Gel stability of Fmoc-Cys and Fmoc-Cys:Fmoc-His was conducted using a vial inversion test, the thermoreversibility test and rheology. Moreover, LC-MS analysis and Ellman’s assay were performed to investigate the Fmoc-Cys oxidation, while fluorescence analysis to reveal critical aggregation concentrations (CAC) of Fmoc-amino acids. The p-NPA assay was used to evaluate the catalytic activity of hydrogels. The results suggest that Fmoc-Cys:Fmoc-His co-assembled hydrogels exhibit superior catalytic activity compared to self-assembled Fmoc-Cys. The lower activity of Fmoc-Cys might be attributed to the formation of disulfide bridges via the oxidation processes, thereby hindering its nucleophilic reactivity. This study contributes to the understanding of low molecular weight hydrogelators and their potential applications as minimalistic catalysts
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