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Porfirini u fotodinamičkoj inaktivaciji mikroorganizama u biofilmu
No full textSposobnost bakterija i gljivica da tvore biofilmove, sesilne zajednice mikroorganizama odgovorne za patogenezu mnogih kroničnih i perzistentnih infekcija, povećava njihovu otpornost na vanjske uvjete i doprinosi razvoju rezistencije na uobičajene antimikrobne agense. Obzirom na alarmantnu situaciju u području liječenja infekcija izazvanih patogenima koji su razvili antimikrobnu rezistenciju, raste potreba za pronalascima novih metoda liječenja. Jedan od mogućih pristupa liječenju infekcija izazvanih mikroorganizmima u biofilmu je primjena fotodinamičke inaktivacije. Fotodinamička inaktivacija mikroorganizama ima nespecifičan mehanizam djelovanja, a zasniva se na međudjelovanju ne-toksičnog fotosenzibilizatora, vidljive svjetlosti i molekularnog kisika, prilikom čega dolazi do generiranja citotoksičnih reaktivnih kisikovih vrsta i singletnog kisika. Većina fotosenzibilizatora zasniva se na tetrapirolnoj strukturi, među kojima su najzastupljeniji porfirini. Neki od porfirinskih spojeva, poput porfimer natrija i endogenog protoporfirina IX, već se primjenjuju kao fotosenzibilizatori u terapiji nekih karcinoma, a sada se razmatra njihova mogućnost upotrebe u inaktivaciji mikroorganizama u biofilmu. Oba su ostvarila značajnu inaktivaciju kod ispitivanih biofilmova. Drugi fotosenzibilizatori su još u eksperimentalnoj fazi te njihovo djelovanje nije dovoljno ispitano. Unatoč značajnoj inaktivaciji postignutoj kod planktonskih mikroorganizama, tetrakationski 5,10,15,20-tetra-(N-metilpiridin-4-il)porfirin tetra-(p-toluensulfonat) nije postigao zadovoljavajuću efikasnost kod većine ispitivanih biofilmova. Dikationski 5,15-bis-[4-(3-trimetilamoniopropiloksi)fenil]porfirin diklorid pokazao se kao efikasan fotosenzibilizator u inaktivaciji gljivičnih biofilmova, no još uvijek nedostaju istraživanja o njegovoj djelotvornosti na biofilmovima bakterijskog podrijetla.The ability of bacteria and fungi to form biofilms, sessile communities of microorganisms responsible for the pathogenesis of many chronic and persistent infections, increases their hardiness to external conditions and contributes to the development of resistance to conventional antimicrobial agents. Given the alarming situation in the field of treating infections caused by pathogens that have developed antimicrobial resistance, there is a growing need for discoveries of new treatment methods. One of the possible approaches to treatment of infections caused by microorganisms in biofilm is the use of photodynamic inactivation. Photodynamic inactivation of microorganisms has a non-specific mechanism of action, based on the interaction of non-toxic photosensitizers, visible light and molecular oxygen, resulting in the generation of cytotoxic reactive oxygen species and singlet oxygen. Most photosensitizers are based on the tetrapyrrolic structure, with a high prevalence on the use of porphyrins. Some porphyrin compounds, such as porfimer sodium and endogenous protoporphyrin IX, are already being used as photosensitizers in the therapy of some cancers, and their potential for inactivation of microorganisms in biofilm is under investigation. Both photosensitizers have caused significant inactivation of the tested biofilms. Other photosensitizers are still in the experimental stage and their activity has not yet been sufficiently studied. Despite the significant inactivation achieved in planktonic microorganisms, tetracationic 5,10,15,20-tetra-(N-methylpyridinium-4-yl) porphyrin tetra-(p-toluenesulfonate) did not achieve satisfactory efficacy in most of the biofilms studied. Dicationic 5,15-bis-[4-(3-trimethylammonio-propyloxy) phenyl]porphyrin dichloride has shown to be an efficient photosensitizer in the inactivation of fungal biofilms but there is still no research on its efficacy in biofilms of bacterial origin
Methods of pyridylporphyrin oxidation and synthesis
No full textPorfirini su organski spojevi koji se mogu koristiti kao fotoosjetljivi spojevi (PS) u fotodinamičkoj terapiji (PDT). PDT uključuje administraciju PS-a u tijelo, gdje se isti lokalizira u tumorskom tkivu te, nakon osvjetljavanja svjetlošću određene valne duljine, uzrokuje odumiranje tumorskog tkiva.
Najčešća metoda sinteze porfirina u našem laboratoriju je modificirana Adler- Longo reakcija koja se relativno jednostavno izvodi, brza je i daje nečistoće koje se mogu pročistiti stupčanom kromatografijom. Zwitterionski oksidirani porfirini imaju malu toksičnost u “mraku”, što je bitno za fotodinamičku terapiju (PDT), u usporedbi sa svojim kationskim analozina, N-metiliranim piridilporfirinima. Stoga, smo naše istraživanje usmjerili na proučavanje upravo ove grupe porfirinskih PS-ova.
Jedan od zahtjeva za porfirine koji se efikasno primjenjuju u PDT, je njihov visoki unos u ciljane stanice. Stoga, N-oksidirani piridilporfirini koji su nam od interesa imaju lipofilnu komponentu, a njihova sinteza u okviru ovog istraživanja uključuje adiciju dugih alkilnih lanaca koji potječu od dodekanoil klorida ili heksadekanoil klorida.
U ovom radu, različite metode N-oksidacije piridilporfirina su analizirane kako bi se istražilo koja metoda rezultira najvećim iskorištenjem oksidiranih porfirina s minimalnom prisutnošću nečistoća za potencijalnu primjenu u PDT-u. Četiri analizirane metode uključuju N-oksidaciju pomoću: meta- kloroperbenzojeve kiseline (m- CPBA); dimetildioksirana (DMD); hodikovog peroksida, natrijeva hidroksida i meta- kloroperbenzojeve kiseline; ili pomoću octene kiseline. Samo su se metode N-oksidacije pomoću m-CPBA i DMD-a pokazale optimalnima, jer su rezultirale visokim iskorištenjem piridilporfirina (65 % za m-CPBA i 70 % za DMD) u reakcijama koje su se lagano mogle optimizirati i izvesti. Metoda koja uključuje upotrebu H2O2, NaOH i m-CPBA, dala je iskorištenje od 3,92% a reakcija s CH3COOH nije dala produkte.
UV/Vis spektroskopska analiza nije pokazala razliku između spektara simetričnog porfirina oksidiranima upotrebom različitih metoda.
Zaključak istraživanja jest kako metoda 1 (m-CPBA) i 2 (DMD) daju zadovoljavajuća iskorištenja. Iako je oksidacija metodom 2 zahtjevnija zbog potrebe za stalnim podešavanjem pH vrijednosti, daje produkt s manje onečišćenja, te je iskorištenje veće za do 5%.Porphyrins are organic compounds that can be used as photosensitizers in photodynamic therapy (PDT). PDT involves photosensitizer (PS) administration into the body, where they penetrate tumour tissues and, once illuminated, cause tumour degradation.
Most common way of porphyrin synthesis in our laboratory includes modified Adler-Longo method, since it is relatively easy to conduct, it is fast and gives us products with impurities separable with a column chromatography. Recently we have found that zwitterionic N-oxidised pyridylporphyrins as PSs in PDT exhibit low “dark” toxicity, as opposed to their cationic analogues, N-methylated pyridylporphyrins, thus we decided to focus our research on this perspective group of porphyrin PSs.
One of the requirements for porphyrins to be used in PDT efficiently, is their high uptake into targeted cells. Therefore, N-oxidised pyridylporphyrins we are particularly interested in have lipophilic component, and their synthesis in this work includes adding long alkyl chains derived from dodecanoyl and hexadecanoyl chlorides.
In this work different methods of pyridylporphyrin N-oxidation were analysed to investigate which method is the best in terms of yields and ease of purification for preparation of new potential photosensitisers for the use in PDT. Four methods were assessed for N-oxidations of porphyrins using: meta-chloroperoxybenzoic acid (m-CPBA), dimethyldioxirane (DMD); hydrogen peroxide, sodium hydroxide and meta-chloroperoxybenzoic acid; or acetic acid. Only methods using m-CPBA and DMD showed promising results, providing high yields of oxidised pyridylporphyrins (65 % for m-CPBA and 70 % for DMD) in the reactions that were easy to optimize and conduct. Method including H2O2, NaOH and m-CPBA gave a yield of (oxydo)pyridylporphyrin of 3,92 % and the reaction using CH3COOH did not occur.
UV/ Vis spectroscopic analysis showed no difference between the spectra of symmetric porphyrin oxidised by the different methods.
We are concluding from this work that both methods 1 (m-CPBA) and 2 (DMD) provide good yields. Although method 2 is more complicated to perform due to constant pH adjustments, the product contains less impurities, needs less additional purification and the yield is up to 5 % higher in comparison to the method 1
Učinak inhibicije fosfatidilinozitol-4 kinaze na adheziju ljudskih trombocita
No full textPlatelets, although for a long time only associated with the process of hemostasis, are now gaining scientific interest because of their emerging roles in processes like the modulation of the immune system, viral infections and cancer. However, platelets’ signaling network is still not completely understood because of the numerous activatory, inhibitory and negative feedback signals all interconnected in a finely regulated system responsible for platelet function and responsiveness to their environment. Platelet cytoskeletal rearrangements during activation are mediated by well-known lipid second messengers, phosphatidylinositol 4,5‐bisphosphate (PIP2) and phosphatidylinositol 3,4,5‐trisphosphate (PIP3), however, the role of phosphatidylinositol 4‐phosphate (PI4P) has not been studied thus far. Therefore, our aim was to investigate the role of phosphatidylinositol 4-kinase (PI4K), an enzyme responsible for producing PI4P, a lipid molecule long thought to be only a precursor for PIP2 and PIP3. Transcriptomic and proteomic data show high levels of several PI4K isoforms in mouse and human platelets. We confirmed the presence of PI4K type II (α and β) in human platelets isolated from blood of several different donors as well as in megakaryocytic cell line DAMI using Western blot analysis. Also, we show the presence of PI4K type II (α and β) by immunostaining, as well as their product, PI4P in human platelets. Finally, we determined that PI4Kα is essential for platelet spreading on glass by specific pharmacological inhibition of PI4Kα. Inhibition of PI4Kα dose dependently decreased F-actin staining, and increased number of platelets with filopodia and actin nodules, characteristic of early platelet activation events. We conclude that PI4Kα is an essential part of the platelet signaling network, specifically the activatory pathways responsible for platelet spreading with roles in modulating actin cytoskeleton.Iako ih se dugo vezalo samo uz pojam hemostaze, trombociti sve više pobuđuju interes znanstvene zajednice zbog otkrića novih uloga koje imaju u procesima poput modulacije rada imunološkog sustava, virusnih infekcija te raka. Međutim, signalna mreža trombocita još uvijek nije u potpunosti razjašnjena. Razlog tome je velik broj aktivacijskih, inhibitornih te negativnih povratnih signala međusobno isprepletenih u fino reguliran sustav koji im omogućuje brzu prilagodbu odgovora na signale iz okoline. U regulaciji promjena citoskeleta tijekom aktivacije trombocita ključna su dva lipidna sekundarna glasnika, fosfatidilinozitol 4,5-bisfosfat (PIP2) i fosfatidilinozitol 3,4,5-trisfosfat (PIP3), međutim uloga fosfatidilinozitol 4-fosfata (PI4P) do sada nije izučavana. Stoga je naš cilj bio istražiti ulogu fosfatidilinozitol 4-kinaze (PI4K), enzima koji proizvodi PI4P, lipidnu molekulu koju se dugo smatralo samo prekursorom PIP2 i PIP3. Transkriptomski i proteomski podaci pokazuju visoku ekpresiju nekoliko izoformi PI4K u mišjim i ljudskim trombocitima. Potvrdili smo ekspresiju PI4K tipa II (α i β) u ljudskim trombocitima različitih donora te megakariocitnoj staničnoj liniji DAMI Western blot analizom. Dodatno, njihovu prisutnost u ljudskim trombocitima, kao i onu njihovog produkta, PI4P-ja, potvrdili smo imunobojanjem. Konačno, specifičnom farmakološkom inhibicijom PI4Kα utvrdili smo da je PI4Kα neophodna za širenje trombocita aktiviranih na staklu. Inhibicija PI4Kα dovela je do smanjenog bojanja F-aktina te povećanog broja trombocita s filopodijima i aktinskim nodulima koji su karakteristični za rane faze aktivacije. Zaljučujemo da je PI4Kα ključan dio signalne mreže trombocita, točnije aktivacijskih putova odgovornih za širenje trombocita te sudjeluje u modulaciji aktinskog citoskeleta
Molekularna dinamika u studijama modulatora aktivnosti γ-sekretaze za liječenje Alzheimerove bolesti
No full textBackground: Alzheimer's disease is the most expensive disease for healthcare providers in developed countries in the 21st century, and the most lucrative market for the pharmaceutical industry. Changes in the activity of the membrane embedded protease γ-secretase can result in a decreased clearance of amyloid proteins and the development of Alzheimer's disease. We analyzed the catalytic mechanism of γ-secretase using some of the best known drug candidates; semagacestat, DAPT, avagacestat, and LY411-575. Molecular docking studies were first used for the identification of binding sites, and multi-scale molecular dynamic simulations were used for the description of binding interactions.
Results: Molecular dynamics studies show that the proteolytic channel of γ-secretase, as well as surface enzyme binding sites, can bind several drug candidates at the same time. There are three surface binding sites that bind ligands over a time period of 300 nanoseconds. At least two ligands can bind inside the open active site tunnel when there is no substrate present. Each step in substrate catalysis has a different affinity for different drug candidates. Transient interactions between positive and negative amino acids in the active site can regulate the catalytic cleavages of amyloid beta precursor protein.
Conclusion: Multiple biphasic modulators can bind to γ-secretase simultaneously and modulate its Aβ production. The biphasic modulators that activate γ-secretase and act as competitive inhibitors could be the most promising drug for Alzheimer's disease. Molecular dynamics can be used to design and develop such drug candidates.Pozadina: Alzheimerova bolest najskuplja je bolest za pružatelje zdravstvenih usluga u razvijenim zemljama u 21. stoljeću.Također je najunosnije tržište za farmaceutsku industriju. Promjene u aktivnosti proteina γ-sekretaze mogu rezultirati smanjenim pročišćavanjem amiloidnih proteina i razvojem Alzheimerove bolesti. Analizirali smo katalitički mehanizam γ-sekretaze koristeći neke od najpoznatijih kandidata za lijekove; semagacestat, DAPT, avagacestat i LY411-575. Studije molekularnog vezanja najprije su korištene za prepoznavanje mjesta vezanja, a za opis interakcija vezanja korištene su simulacije molekularne dinamike.
Rezultati: Studije molekularne dinamike pokazuju da proteolitički kanal γ-sekretaze, kao i mjesta vezanja na enzimskoj površini, mogu istovremeno vezati nekoliko kandidata za lijek. Postoje tri površinska mjesta vezivanja koja vežu ligande u vremenskom periodu od 300 nanosekundi. Barem dva liganda mogu se vezati unutar otvorenog tunela aktivnog mjesta kada nema supstrata. Svaki korak katalize supstrata ima različit afinitet za različite kandidate za lijek. Privremene interakcije između pozitivnih i negativnih aminokiselina na aktivnom mjestu mogu regulirati katalitičko cijepanje proteina prekursora amiloida beta.
Zaključak: Višestruki dvofazni modulatori mogu se istovremeno vezati za γ-sekretazu i modulirati njenu proizvodnju Aβ. Dvofazni modulatori koji aktiviraju γ-sekretazu i djeluju kao kompetitivni inhibitori mogli bi biti najperspektivniji lijek za Alzheimerovu bolest. Molekularna dinamika može se koristiti za oblikovanje i razvoj takvih kandidata za lijekove
Chemoresistance in colon cancer: focus on conventional chemotherapy
No full textColorectal cancer (CRC) is a widespread tumour type amongst men and women. Despite the available screening tests, this disease is most likely to be discovered when already having progressed to an advanced stage that is palliatively treated with conventional or cytotoxic chemotherapeutics. The latter typically involves 5-fluorouracil (5-FU), oxaliplatin (Ox) and irinotecan (CPT-11), first-line therapeutics that stumble upon chemoresistance in the majority of cases of metastatic or advanced CRC.
This study aims to integrate the results derived from different studies in CRC cancer, found on PubMed, and finally to bioinformatically process them in order to identify some novel molecular features of drug resistance that could be specifically targeted in order to sensitize CRC to conventional chemotherapy. The first step was to analyse the complex interactions between biomarkes of chemoresistance to 5-FU, Ox and CPT-11 with the help of STRING. The STRING-derived PPI network was then imported in cytoHubba to yield hub genes based on their number of interactions. Enrichments in DAVID biological processes, cell compartments and molecular functions as well as enrichments in KEGG pathways were also investigated.
As a result, newly identified hub genes were yielded for the chemoresistance to 5-FU (RPL8, RPS18, RPS11), Ox (ERCC3, ERCC4) and CPT-11 (CTNNB1, CTNND1). Also, pathways in common to the resistance to all three drugs were determined: miRNAs in cancer, proteoglycans in cancer, cellular senescence and the sphingolipid signalling pathway. Targeting the hub genes and signalling pathways proposed in this review might be effective against chemoresistance to conventional chemotherapy.Karcinom debelog crijeva učestala je zloćudna bolest koja pogađa oba spola. Najčešće se otkrije tek u trećoj ili četvrtoj fazi, a tada se palijativno tretira citotoksičnim kemoterapeuticima-5-fluorouracilom (5-FU), oksaliplatinom (Ox), irinotekanom (CPT-11). Učinak navedenih lijekova smanjuje se radi razvoja kemorezistencije, koja je ujedno i najčešći ishod dugotrajnijeg tretiranja napredne i metastatske faze karcinoma debelog crijeva.
Svrha ovog revijalnog rada bila je sažimanje rezultata znanstvenih radova s naglaskom na mehanizme kemorezistencije u kolorektalnom karcinomu. Znanstveni radovi pretraženi su putem online baze podataka PubMed, a izdvojeni biomarkeri kemorezistencije zatim su bioinformatički analizirani. Bioinformatičkom analizom identificirale su se nove molekularne značajke kemorezistencije na koje bi se moglo farmakološki djelovati u svrhu pojačavanja odgovora kolorektalnog carcinoma na 5-FU, Ox i CPT-11.
Platformom STRING dobivene su proteinske interakcijske mreže, iz kojih su programom cytoHubba izdvojeni hub geni ključni za kemorezistenciju na 5-FU, Ox te CPT-11. Zatim, programima DAVID i KEGG odredili su se biološki procesi, dijelovi stanice, molekulske funkcije te signalizacijski putevi u kojima sudjeluju istraženi biomarkeri .
Na kraju, novoidentificirani su hub geni koji bi mogli igrati ulogu u kemorezistenciji na 5-FU (RPL8, RPS18, RPS11), Ox (ERCC3, ERCC4) i CPT-11 (CTNNB1, CTNND1 te signalizacijski putevi zastupljeni u kemorezistenciji na sva tri lijeka: miRNA i proteoglikani u raku, stanična senescencija te signalni put sfingolipida. Ciljanje novoidentificiranih hub gena i signalizacijskih puteva, moglo bi se pokazati uspješnim protiv kemorezistencije na konvencionalnu kemoterapiju.
Ključne riječi: karcinom debelog crijeva, 5-fluorouracil, oxaliplatin, irinotecan, kemorezistencij
Effects od additional feeding of honey bee colonies (Apis mellifera) with probiotic cultures
No full textZajednice medonosne pčele (Apis mellifera L.) smatraju se ekonomski najvrjednijim oprašivačima biljaka te na taj način izravno utječu na proizvodnju voća i povrća za prehranu ljudi. U novije vrijeme zdravlje pčela sve je više narušeno, što se vidi po učestalim uginućima pčelinjih zajednica. Uzroci ugibanja pčelinjih zajednica mogu biti različiti, a najviše se spominju bolesti i nepovoljni utjecaj primjene pesticida u intenzivnoj poljoprivrednoj proizvodnji. Razvojem modernih molekularnih metoda upoznat je sastav i važnost crijevne mikrobiote za imunitet i zdravlje pčela. Uočeno je da pčele koje su u stanju „stresa“ ili pate od neke bolesti ujedno imaju narušen sastav crjevne mikrobiote. Zbog korelacije između sastava mikrobiote i zdravlja pčelnjih zajednica, počelo se istraživati potencijalne probiotičke bakterije na pčelama. Nekoliko prethodnih istraživanja su pokazala kako se pomoću bakterija roda Lactobacillus dodanih u prehranu može postići povoljan utjecaj na zdravlje pčelinje zajednice.
Iz probavnog sustava pčela izolirane su kulture bakterija iz roda Lactobacillus. Izolirane bakterije umnožene su u MRS tekućem hranjivom mediju, a zatim su u obliku probiotičkog šećernog sirupa dodane pčelinjim zajednicama. Mjerenjem površine pčelinjeg legla na saću u različitim terminima uočeno je kako su pčelinje zajednice tretirane probioticima u usporedbi s kontrolnim brže napredovale. Brojanjem spora Nosema spp. u crijevima uzorkovanih pčela svjetlosnim mikroskopom utvrđeno je da su zajednice tretirane probioticima imale smanjeni stupanj invazije nozemozom tijekom pčelarske sezone. In vitro pokusima pokazali smo kako probiotičke bakterije usporavaju rast bakterije Paenibacillus larvae koja je uzročnik jedne od najopasnijih bolesti pčelinjih zajednica – američka gnjiloća medonosne pčele. Inhibicija rasta P. larvae in vitro ukazuje kako probiotičke bakterije izlučuju metabolite koji sprječavaju rast patogenih mikroorganizama. Rezultati ovog istraživanja potvrđuju neke od prethodno objavljenih rezultata, no za bolje razumijevanje mehanizama djelovanja probiotika na pčele potrebno je provesti in vitro pokuse i na drugim uzročnicima bolesti koji nisu bili predmet ovog istraživanja. Za potrebe razvoja i kliničkih istraživanja probiotika za pčele potrebno je provesti pokuse sa više sojeva bakterija i na većem broju pčelinjih zajednica.The honey bee (Apis mellifera L.) is considered to be the most economically valuable pollinator of plants and thus directly affecting the production of fruits and vegetables for human consumption. In recent times, the health of bees has been increasingly impaired, as can be seen by the frequent deaths of the honeybee colonies. The causes of bee community deaths may vary, with the greatest mention of diseases and the adverse impact of pesticide use in intensive agricultural production. With the development of modern molecular methods, the composition and importance of the intestinal microbiota on the immunity and health of the honey bees are introduced. It has been observed that bees that are in a state of "stress" or suffering from a disease also have impaired composition of the gut microbiota. Because of the correlation between microbiota composition and health of the honey bees, an investigation of potential probiotic bacteria has been started. Several previous studies have shown that bacteria from the genus Lactobacillus added to the diet can have a beneficial effect on the health of the honeybee colony.
Bacteria from the genus Lactobacillus were isolated from the digestive system of the honey bees. Isolated bacteria were multiplied in MRS broth and then added to bee colonies in the form of a probiotic sugar syrup. By measuring the surface of brood in different terms, it was observed that the honeybee colonies treated with probiotics progressed faster compared to controls. By counting the Nosema spp. spores in the intestines of sampled bees with optic microscope, it was found that probiotic-treated colonies had a reduced degree of nosemosis invasion during the beekeeping season. In vitro experiments have shown that probiotic bacteria slow down the growth of Paenibacillus larvae, which is cause of one of the most dangerous diseases of the honeybee colonies – American foulbrood. Inhibition of the growth of P. Larvae indicates that probiotic bacteria excrete metabolites which prevent growth of pathogen microorganisms. The results of this study confirm some of the previously reported results. In order to gain a better understanding of the mechanisms of action of probiotics on the honey bees, in vitro experiments with more honey bee pathogens need to be done. In order to conduct clinical studies and develop probiotic for the honey bees, experiments with more bacterial strains and on more honeybee colonies need to be conducted
Rational design, synthesis and biological profiling of new JMJD2C inhibitors
No full textJMJD2 je enzimska obitelj humanih histon demetilaza koje su povezane s bolestima kao što su karcinom dojke i prostate. Ti enzimi smatraju se onkogenima i njihova selektivna inhibicija mogla bi biti pristup za tretiranje spomenutih karcinoma. Literaturno poznati inhibitori imaju nisku staničnu permeabilnost i selektivnost, zbog čega su daljnja istraživanja usmjerena napoboljšanje aktivnosti, selektivnosti i ADME svojstava.
U ovom istraživanju korišteni su dostupni literaturni podaci o poznatim inhibitorima kao kristalne strukture enzima JMJD2A/JMJD2C u kompleksu s malim molekulama. Analizom literaturno dostupnih kristalnih struktura proučene su interakcije između proteina i liganda te su metodama molekulskog modeliranja dizajnirani i zatim sintetizirani novi potencijalni inhibitori s toksoflavinskim strukturnim motivom.
Kroz biološko profiliranje supstanci u in vitro pokusima određena je njihova inhibicijska aktivnost na JMJD2C enzimu, zatim stanična permeabilnost i metabolička stabilnost molekula, te utjecaj na viabilnost stanica.JMJD2 is human histone demethylase family of enzimes that are linked to diseases such as prostate and breast cancer. These enzymes are considered oncogenes and their selective inhibition might be a therapeutic approach to treat the cancer. Currently known inhibitors have low permeability and low selectivity, so the further investigation is focused on optimizing activity, selectivity and ADME data.
In my thesis I used available literature data on known inhibitors and x-ray structures of JMJD2C complexes with small molecules. A computational analysis of protein-ligand interactions was applied using structure based drug design tools to design potential JMJD2C inhibitors with a toxoflavin motif. Chemical synthesis of designed compounds was developed.
New compounds were profiled in vitro on JMJD2C enzyme inhibition, membrane permeability and microsomal stability as well as their influence on cell viability
Spinal cord neural stem cells heterogeneity in postnatal development
No full textNeural stem cells are capable of generating new neurons during development as well as in the adulthood and represent one of the most promising tools to replace lost or damaged neurons after injury or neurodegenerative disease. Unlike the brain, neurogenesis in the adult spinal cord is poorly explored and the comprehensive characterization of the cells that constitute stem cell neurogenic niche is still missing. Moreover, the terminology used to specify developmental and/or anatomical CNS regions, where neurogenesis in the spinal cord occurs, is not consensual and the analogy with the brain is often unclear. In this review, we will try to describe the heterogeneity of the stem cell types in the spinal cord ependymal zone, based on their origin and stem cell potential. We will also consider specific animal in vitro models that could be useful to identify “the right” stem cell candidate for cell replacement therapies
Charge and size of the self-assembled amphiphilic dye aggregates: An experimental study
No full textSamonakupljanje (asocijacija) individualnih molekula u supramolekularne strukture spontan je proces koji se odvija kao posljedica slabih, nekovalentnih međumolekulskih interakcija. Udruživanje amfifilnih organskih boja u posljednje je vrijeme privuklo značajnu pažnju kako u biološkim tako i u znanostima o funkcionalnim materijalima zahvaljujući njihovim jedinstvenim fotofizikalnim svojstvima. Takvi spojevi najviše se koriste u industrijskim procesima (proizvodnja solarnih ćelija, pročišćavanje otpadnih voda), farmaceutici (razvoj formulacije) te biomedicinskim istraživanjima (prijenos lijekova, oslikavanje). Stjecanje informacija i znanja o procesu samonakupljanja molekula pruža jedinstvene mogućnosti u dizajnu novih materijala za naprednu primjenu u nanotehnologiji.
U ovome radu sintetizirani su jednostavnim supstitucijskim reakcijama amfifilni derivati dviju organskih boja, rodamina B i orange-II. Sintetiziranim spojevima po prvi put je ispitano koloidno ponašanje u vodenom mediju u strogo definiranom koncentracijskom rasponu pri variranim vrijednostima pH i ionske jakosti disperzija. Promjena fotofizikalnih svojstava istražena je spektrofotometrijskim eksperimentom, a karakteristika asocijacije ispitivanih spojeva tehnikama raspršenja svjetla i analizom viseće kapi.
Promjena apsorpcijskih spektara ukazala je na gubitak protonirajućih skupina kod derivata boja te neovisnost veličine samonakupina o pH u blago kiselim iili baznim uvjetima. Mjerenjem hidrodinamičkog promjera i elektroforetske pokretljivosti utvrđeno je da amfifilni derivati boja formiraju vezikule čija veličina varira s koncentracijom i ionskom jakosti. Rezultati mjerenja površinske napetosti na granici faza voda/zrak ukazuju na izrazitu površinsku aktivnost boja uz jasno definiranu vrijednost kritične koncentracije samonakupljanja. Ponavljanjem eksperimenta nakon određenog vremenskog razdoblja ustanovljeno je da se vezikule sačinjene
od sintetiziranih spojeva razlikuju u stabilnosti, odnosno da rodaminskom derivatu treba duže vrijeme da se prilagodi veličinom i oblikom u stabilne strukture.Self-assembly (association) of individual molecules into supramolecular structures is a spontaneous process that takes place as a result of weak, non-covalent intermolecular interactions. The association of amphiphilic organic dyes has attracted considerable attention both in biological and functional materials sciences thanks to their unique photophysical properties. Such compounds are widely used in industrial processes (solar cell production, waste water purification), pharmaceuticals (development of formulations) and biomedical research (drug delivery, imaging). Acquiring information and knowledge about the self-assembly process provides unique opportunities in designing new materials for advanced application in nanotechnology.
In this work, amphiphilic derivatives of two organic dyes, rhodamine B and orange-II are synthesized by simple substitution reactions. For the first time colloid behaviour of synthesized compounds in aqueous media was examined in a strictly defined concentration range at varied pH and ionic strength values of dispersion. The change of photophysical properties was investigated in a spectrophotometric experiment, while the association of the tested compounds by means of light scattering techniques and pendant drop analysis.
The change of the absorption spectra indicated loss of free ionizable groups for the amphiphilic dye derivatives and independence of their self-assembly on pH in mildly acidic and basic conditions. By measuring the hydrodynamic diameter and the electrophoretic mobility, the derivatives were found to form vesicles whose size varies with concentration and ionic strength. The results of the surface tension measurement indicate a pronounced surface activity as well as clearly defined critical concentration of self-assembly. By repeating the experiment over a time period, it was shown that the studied compounds differ in stability, in particular the rhodamine derivative takes longer to adapt a stable structure with a well defined size and shape
Testing the role of L-type Cryptochrome in circadian and circalunar light dependent behavior, in the marine bristle worm Platynereis dumerilii
No full textIn order to maximize their chances of survival and procreation animals have to be able to predict and adapt to a range of periodic changes in their environment. To be able to anticipate environmental rhythms, like day-night illumination, they developed an endogenous timekeeping oscillators (circadian clocks), with approximately the same period length and phase as the external cycle they are subjected to. Endogenous clocks use external cues to entrain to the environment and produce behavioral and physiological outputs to help animals adjust accordingly. Light is the most prominent entrainment cue (zeitgeber) and light-responsive cryptochromes have been proven to facilitate entrainment in species like Arabidopsis thaliana, Soganus luridus and Drosophila melanogaster.
Since marine habitats harbor a multitude of natural rhythms, marine organisms often have more than one type of clock. Marine annelid Platynereis dumerilii possess two clocks entrained by light: circadian clock, controlling their daily locomotor activity, and circalunar clock, controlling the timing of their sexual maturation. Considering P. dumerilii possess a light responsive Drosophila-type cryptochrome (L-cry), I hypothesized L-cry acts as a putative blue-light receptor facilitating clock function, so loss-of-function animals (L-cry-/-) should display disruptions in circalunar and circadian behavioral and physiological outputs.
I tested the hypothesis by performing a maturation pattern analysis to see how L-cry influences the circalunar rhythm, and multiple circadian experiments to test its circadian role. To confirm unpublished laboratory observations of L-cry-/- animals developing slower than their L-cry+/+ and L-cry+/- siblings I also performed a regeneration experiment. The experiments showed L-cry-/- exhibit a major maturation delay likely due to overall developmental delay especially in segment addition, regeneration and gametogenesis. L-cry likely does not play a dominant role in maturation
itself, but mechanisms involved could be under circalunar or circadian influence. L-cry-/- also exhibited changes in timing of circalunar maturation as well as increase in arrhythmic circadian behavior and susceptibility to masking effects of light, which confirms my hypothesis that L-cry has a crucial role in circadian and circalunar clocks of P. dumerilii.Kako bi si povećali izglede za preživljavanje i reprodukciju životinje trebaju predvidjeti i prilagoditi se nizu periodičkih promjena u svom okruženju. Da bi mogle predosjetiti prirodne ritmove, poput promjena intenziteta svjetlosti tijekom dana i noći, razvile su endogene oscilatore za mjerenje vremena (cirkadijalne satove), koji imaju približno jednaki period i fazu kao i vanjski ciklus pod čijim su utjecajem. Biološki sat koristi signale iz okoline kako bi se sinkronizirao s uvjetima okoliša te uzrokuje fiziološke i bihevioralne promjene u životinja kao odgovor. Svjetlost je najjači okolišni signal koji može sinkronizirati endogene satove (zeitgeber signal), a kriptokromi osjetljivi na svjetlost dokazano omogućuju sinkronizaciju bioloških satova putem svjetla u vrsta poput Arabidopsis thaliana, Soganus luridus i Drosophila melanogaster.
S obzirom da su morska staništa pod utjecajem niza prirodnih ciklusa, morski organizmi često imaju više vrsta bioloških satova. Morski kolutićavac Platynereis dumerilii ima dva biološka sata pod utjecajem svjetla: cirkadijalni sat, koji kontrolira lokomociju tokom dana i noći, te circalunarni sat, koji kontrolira temporalni aspekt spolnog sazrijevanja. S obzirom da P. dumerilii posjeduje tip kriptokroma srodan onom kod Drosophilae (L-cry) postavila sam hipotezu prema kojoj je L-cry potencijalni receptor plavog svjetla koji sudjeluje u radu bioloških satova. Prema hipotezi L-cry-/- mutanti, bez funkcionalnog L-Cry proteina, trebali bi pokazati poremećaje u cirkadijalnom i cirkadijalnom ponašanju te povezanoj fiziologiji.
U svrhu testiranja hipoteze provela sam analizu obrasca spolnog sazrijevanja kako bi istražila utjecaj L-cry gena na cirkalunarni sat te niz cirkadijalnih eksperimenata, kako bi istražila njegovu ulogu u cirkadijalnom ponašanju. Kako bi potvrdila neobjavljena zapažanja unutar laboratorija prema kojima se L-cry-/- životinje razvijaju sporije od L-cry+/+ i L-cry+/- potomaka provela sam analizu sposobnosti regeneracije. Eksperimenti su
pokazali da L-cry-/- jedinke sazrijevaju znatno kasnije, vjerojatno zbog generalnog zastoja u razvoju, u fazi dodavanja segmenata, regenereacije i gametogeneze. L-cry vjerojatno nema dominantnu ulogu u samom procesu spolnog sazrijevanja, ali uključeni mehanizmi mogu biti pod utjecajem cirkalunarnog ili cirkadijalnog sata. L-cry-/- jedinke su također pokazale promjene u tempiranju cirkalunarnog sazrijevanja, kao i povećanje u aritmičnom cirkadijalnom ponašanju i podložnosti „masking“ efektu svjetlosti, što potvrđuje moju hipotezu da L-cry ima ključnu ulogu u cikalunarnim i cirkadijalnim satovima P. dumerilii