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Istraživanje koagregacije EHD3 i njegove potencijalne uloge u kroničnim mentalnim bolestima
No full textChronic mental illnesses (CMI) affect people’s daily lives including their thinking, functioning and emotions. These include schizophrenia, bipolar disorder and major depressive disorder, and are characterized by their perpetual and recurrent nature. There has therefore been a need to identify possible biological targets to get a deeper understanding of mental illnesses and to improve treatment and diagnosis standards. One newly studied concept is protein aggregation as a possible non-genetic cause of chronic mental illnesses. Five proteins were previously discovered to aggregate in brains of patients with CMI. Some of these have been confirmed to aggregate on their own, with a suggestion that each of them might influence the representation of a specific subtype of previously mentioned chronic mental illnesses. However, there have also been instances of co-aggregation in which case one protein stimulates the other one to aggregate. Co-aggregation represents the possibility that some or all of these proteins may interact with each other as a part of a single aggregation syndrome.
EHD3 is a newly discovered aggregating protein in mental illness, a member of the EHD protein family, encoded by the EH domain-containing 3 gene. While there is not a lot known about its functions, it is believed to be one of the key regulators of endocytic transport and involved in promotion of dopaminergic transmission. In this research, we investigated the aggregation properties of EHD3 and specifically possible co-aggregation of EHD3 with TRIOBP-1 and Dysbindin 1A in mammalian cells. TRIOBP-1 is an aggregating protein involved in modulation of actin through binding to F-actin fibers, while Dysbindin 1A is involved in regulation of neurotransmitter release and brain development.
Fluorescent microscope results have shown that while EHD3 aggregated both alone and in the presence of the two proteins, there was no sign of co-aggregation. Future research should focus on investigating both whether EHD3 aggregates in cells with other proteins implicated in mental illnesses, but also on whether aggregation of EHD3 and these other proteins occurs in the same patients.Kronične mentalne bolesti utječu na svakodnevne živote ljudi, uključujući njihovo razmišljanje, funkcioniranje i osjećaje. Njima pripada shizofrenija, bipolarni poremećaj i klinička depresija, a karakterizira ih trajnost i ponovno javljanje. S obzirom na to, postoji potreba za pronalaskom mogućih bioloških meta kako bismo ostvarili dublje razumijevanje mentalnih bolesti i unaprijedili standarde dijagnoze i tretmana. Agregacija proteina kao mogući ne-genetski uzročnik kroničnih mentalnih bolesti jedan je od novije istraživanih pojmova u ovom području. Prethodno je otkriveno pet proteina koji agregiraju u takvim bolestima. Za neke od njih potvrđeno je da agregiraju samostalno, pri čemu svaki zasebno može utjecati na prezentaciju određenih podvrsta prethodno navedenih bolesti. Međutim, zabilježeni su i slučajevi kada jedan protein potiče drugog na agregiranje. Koagregacija predstavlja mogućnost da neki ili svi navedeni proteini međudjeluju zajedno kao dio agregacijskog sindroma.
EHD3 je novootkriveni agregirajući protein u mentalnim bolestima. Dio je proteinske obitelji EHD, a kodiran je od strane gena koji sadrži EH domenu 3. Iako se trenutno ne zna mnogo o njegovoj ulozi, smatra se da je jedan od ključnih regulatora endocitoznog prijenosa i da je uključen u promociju transmisije dopamina. U ovom istraživanju, ispitana su agregacijska svojstva EHD3 i potencijalna mogućnost koagregacije EHD3 s proteinima TRIOBP-1 i Dysbindin 1A u stanicama sisavaca. TRIOBP-1 je agregirajući protein uključen u modulaciju aktina vezanjem za F-aktinska vlakna; dok je Dysbindin 1A uključen u regulaciju otpuštanja neurotransmitera i razvoj mozga.
Rezultati fluorescentne mikroskopije pokazali su da, iako EHD3 agregira i samostalno i u prisustvu ova dva proteina, nema znakova koagregacije. Buduća istraživanja trebala bi ispitati agregira li EHD3 u stanicama s drugim proteinima povezanim s mentalnim bolestima, kao i pitanje odvija li se agregacija EHD3 i tih proteina kod istih pacijenata
Toxicity of 3-pyridylporphyrins in an in vivo model of Drosophila melanogaster
No full textPorfirini su skupina organskih spojeva, koja se posljednjih godina sve više koristi kao fotosenzibilizator (PS) u fotodinamičkoj terapiji (PDT). PDT se bazira na mehanizmu fototoksične ekscitacije PS-a uz pomoć svjetlosti prikladne valne duljine, čime nastaju reaktivne kisikove vrste odgovorne za citotoksičnost. U pretkliničkim istraživanjima korištenjem in vitro i in vivo pristupa potrebno je dokazati fototoksičnost PS-a samo u prisustvu ekscitacijske valne duljine, kao i netoksičnost u odsutstvu svjetla. Ovaj diplomski rad nastavak je istraživanja (foto)toksičnosti novosintetiziranih N-oksidiranih i N-metiliranih (3-piridil)porfirina, provedenih in vitro na humanim stanicama karcinoma. Cilj rada je razviti protokol ispitivanja stabilnosti i toksičnosti N-oksidiranih i N-metiliranih (3-piridil)porfirina u in vivo modelu D. melanogaster, koristeći TMPyP3. Spektroskopskim metodama potvrđena je kemijska stabilnost TMPyP3 u metanolu, destiliranoj vodi, PBS-u te otopinama proteina i fizioloških kationa. Utvrđeno je kako TMPyP3 ostaje kemijski stabilan samo u hranjivom mediju za D. melanogaster bez dodatka kvasca. Koncentracija apsorbiranog spoja iznosi oko 1nM, a veća je u prethodno izgladnjelim jedinkama hranjenim 180 min s 40 μM TMPyP3 u usporedbi s jedinkama koje nisu izgladnjele, a hranjene su 24 sata. TMPyP3 nije se pokazao akutno toksičnim u mraku za jedinke D. melanogaster. Pokazano je kako PDT LED izvorom crvene svjetlosti pri intenzitetu 40 mW/cm2 nije letalna, ali je potencijalno neurotoksična za D. melanogaster hranjene 40 μM TMPyP3 180 min neovisno o trajanju osvjetljavanja. Protokol ispitan ovim radom pokazao se efikasnim za detekciju fototoksičnosti PDT terapije 3-piridilporfirinima u in vivo modelu D. melanogaster. Daljnjim razvojem protokola, D. melanogaster mogla bi postati važan model u pretkliničkim studijama 3-piridilporfirina, omogućujući brzo i ekonomično testiranje toksičnosti velikog broja ovih spojeva, nakon čega će samo najbolji kandidati pristupiti klasičnim studijama na modelima sisavaca.Porphyrins are a group of organic compounds, which have been increasingly used as photosensitizers (PS) in photodynamic therapy (PDT) in recent years. PDT is based on the mechanism of phototoxic excitation of PS with the help of light of appropriate wavelength, thus producing reactive oxygen species responsible for cytotoxicity. In preclinical studies using the in vitro and in vivo approaches, it is necessary to demonstrate the phototoxicity of PS only in the presence of excitation wavelength, as well as non-toxicity in the absence of light. This thesis is a continuation of the (photo)toxicity study of newly synthesized N-oxidized and N-methylated (3-pyridyl)porphyrins, conducted in vitro on human cancer cells. The aim of this study was to develop a protocol for testing the stability and toxicity of N-oxidized and N-methylated (3-pyridyl) porphyrins in an in vivo model of D. melanogaster, using TMPyP3. Spectroscopic methods confirmed the chemical stability of TMPyP3 in methanol, distilled water, PBS and solutions of proteins and physiological cations. TMPyP3 was found to remain chemically stable only in nutrient medium for D. melanogaster without yeast addition. The concentration of the absorbed compound is about 1 nM, and is higher in pre-starved flies fed for 180 minutes with 40 μM TMPyP3 compared to non-starved flies fed for 24 hours. TMPyP3 showed no acute dark toxicity for D. melanogaster. It has been shown that the PDT therapy using LED red light source at an intensity of 40 mW/cm2 is not lethal, but is potentially neurotoxic to D. melanogaster fed with 40 μM TMPyP3 for 180 minutes regardless of the duration of illumination. The protocol examined in this study proved effective for detecting the phototoxicity of PDT therapy with 3-pyridylporphyrins in an in vivo model of D. melanogaster. With further development of the protocol, D. melanogaster could become an important model in preclinical studies of 3-pyridylporphyrins, enabling rapid and cost-effective toxicity testing of a large number of these compounds, after which only the best candidates will access classical studies in mammalian models
Utjecaj cirkadijalnih gena na redoks ravnotežu i antioksidativni sustav enzima
No full textReaktivni kisikovi spojevi (ROS) su molekule koje nastaju reakcijama djelomične razgradnje kisika, a karakterizira ih velik oksidacijski potencijal. ROS u stanici se najčešće generiraju kao produkt mitohondrijskog metabolizma ili pod utjecajem vanjskih faktora. Stanje u kojem se stanica nalazi kada se ravnoteža proizvodnje i uklanjanja ROS poremeti naziva se oksidacijski stres. Iako je povećana količina ROS štetna za organizam i povezana s brojnim patološkim stanjima, ROS igraju ključnu ulogu u pravilnom funkcioniranju stanice kao signalne molekule u brojnim fiziološkim procesima. Jedan od načina obrane stanice od oksidativnog stresa je antioksidativni sustav enzima među kojima se posebno ističu katalaza (CAT) i superoksid dismutaza (SOD). Novija istraživanja stavljaju naglasak na cirkadijalnu regulaciju količine ROS iako sam molekularni mehanizam interakcije cirkadijalnih gena i antioksidativnog sustava enzima nije jasno utvrđen.
U provedenom istraživanju ispitivali smo utjecaj cirkadijalnih gena na količinu ROS u modelnom organizmu D. melanogaster na indirektan način, mjerenjem aktivnosti CAT i SOD kod wt mušica te različitih cirkadijalnih mutanata (per01,tim01,ClkJrk i cyc01). Uspoređivali smo aktivnost tih enzima u različito doba dana te razliku aktivnosti u tijelima u odnosu na glave mušica.
Dobiveni rezultati upućuju na postojanje vremenski uvjetovane ekspresije gena koji kodiraju za antioksidative enzime, a regulorani su cirkadijalnim satom. Također, uočljiva je razila u količini aktivnosti CAT i SOD u glavama u odnosu na tijelo mušica potvrđujući tezu kako postoji djelomična autonomija perifernih tkiva u odnosnu na središnji regulatorni sat smješten u suprahijazmatskoj jezgri (SCN). Značajne su bile i razlike aktivnosti enzima među pojedinim genotipovima.Reactive oxygen species (ROS) are molecules produced by reactions of partial oxygen degeneration and are characterized by high oxidative potential. ROS in a cell are usually generated by external factors or as a product of mitochondrial metabolism. The condition in which more ROS are produced than scavenged is called oxydative stress. Although a high level of ROS is damaging for cells and is linked to many pathological conditions, ROS play a crucial role in cell signaling pathways and are necessary for maintaining homeostasis. One of the ways how a cell defends itself from oxidative stress is the antioxidant enzyme system. Some of the most important enzymes of this system are catalase (CAT) and superoxide dismutase (SOD). Recent researches have been focusing on the circadian regulation of ROS levels even though the molecular mechanism of interaction between the circadian genes and the antioxidant enzyme system is still not fully understood.
In the conducted experiment we investigated the impact that circadian genes have on the level of ROS using a model organism D.melanogaster, indirectly, by measuring the activity of CAT and SOD in wild type flies and different circadian mutants (per01,tim01,ClkJrk and cyc01). We have compared the activity of those enzymes during different times of the day and the difference between bodies and heads of flies.
We found that the expression of genes coding for antioxidant enzymes were affected by the time of the day and regulated by the circadian clock. Furthermore, there was a notable difference between CAT and SOD activity in heads and bodies of the flies, thus confirming the existence of partial regulatory autonomy of peripheral tissues compared to the central regulatory clock in suprachiasmatic nucleus (SCN). Differences between the mutants were also significant
Organochlorine compounds in fish from Adriatic sea
No full textPostojani organski spojevi (POPs) su spojevi od globalnog značaja jer značajno utječu na okoliš i ljudsko zdravlje. Razine i raspodjela 17 pojedinačnih kongenera PCB-a i 7 organoklorovih pesticida su istraživane u mišićnom tkivu srdele ulovljene u Jadranskom moru. Svi analizirani spojevi su pronađeni u uzorcima srdela. Indikatorski PCB-153 bio je najzastupljeniji kongener PCB-a. Također su bili dominantni kongeneri PCB-138 i PCB-180. Raspon masenih udjela indikatorskih kongenera PCB-a bio je od 0.06 ng g-1 do 3.29 ng g-1. Raspon toksikološki značajnih PCB-a bio je od 0.02 ng g-1 do 1.16 ng g-1. Raspon masenih udjela OCP-a u srdeli bio je od 0.02 ng g-1 do 2.32 ng g-1. Najdominantniji pesticid u uzrocima bio je p,p'-DDE. Statističkom analizom podataka nisu pronađene značajne razlike u razinama onečišćenja između grupa uzoraka. Rezultati istraživanja su pokazali da su srdele sigurne za konzumaciju na temelju toksikološki relevantnih parametara.Persistent organic pollutants (POPs) are compounds of global importance because they have a significant impact on the environment and human health. The levels and distribution of 17 individual PCB congeners and 7 organochlorine pesticides were investigated in the muscle tissue of sardines caught in the Adriatic Sea. All the compounds analyzed were found in sardine samples. Indicator PCB-153 was the most dominant PCB congener. PCB-138 and PCB-180 congeners were also dominant. The range of PCB indicator congeners was from 0.06 ng g-1 to 3.29 ng g-1. The range of toxicologically significant PCBs was from 0.02 ng g-1 to 1.16 ng g-1. The range of the OCP sardine was from 0.02 ng g-1 to 2.32 ng g-1. The most dominant pesticide in samples was p,p'-DDE. Statistical analysis of the data did not reveal significant differences in contamination levels between the sample groups. Based on toxicologically relevant parameters our results have shown that sardines are safe for consumption
Macrolide conjugates: structure, mechanism of action and biological effects
No full textDugi niz godina makrolidi su bili prvi izbor antibiotika u liječenju bakterijskih infekcija. Međutim, prekomjernom upotrebom antibiotika mnogi bakterijski sojevi razvili su otpornost na primjenjive antibiotike, problem kojim se i danas suočavamo. Makrolidi su bili djelotvorni antibiotici na patogene najčešće respiratornih bolesti, ali danas njihova djelotvornost inhibicije sinteze proteina nije više na istoj razini. No, primijećeno je kako makrolidi u kombinaciji s drugom aktivnom molekulom ostvaruju izvrsno antibakterijsko djelovanje. Time je došlo do razvoja ideje za sintezom molekule s dvostrukim djelovanjem koja bi se sastojala od makrolidne okosnice za koju su vezane različite druge farmakoforne skupine. Nekim od najučinkovitijih bočnih lanaca vezanih za makrolidni prsten pokazali su se kinoloni (makroloni), acilidi, nesteroidni protuupalni lijekovi (NSAID), steroidi i glikokonjugati. U ovom završnom radu opisane su molekule pronađene pretraživanjem literature, a koje posjeduju antibakterijsko i/ili protuupalno djelovanje. Duljina poveznice između dvije molekule je značajan čimbenik antibakterijskog učinka. Najveća antibakterijska učinkovitost postiže se modifikacijom kladinoznog šećera ili modifikacijom u C-3 položaju makrocikičkog prstena. Hibridi makrolida i steroida/NSAID pokazali su poboljšano djelovanje uz specifičniju aktivnost zahvaljujući poboljšanoj akumulaciji unutar upalnih stanica. Kod glikokonjugata je ipak potvrđen smanjen antibakterijski učinak disaharida u odnosu na monosaharide pri čemu veliku ulogu igraju navodno steričke smetnje.For many years, macrolides were the first choice of antibiotics in the treatment of bacterial infections. However, with the overuse of antibiotics, many bacterial strains have developed resistance to applicable antibiotics, a problem we still face today. Macrolides were effective antibiotics on the pathogens of most common respiratory diseases, but today their effectiveness in inhibiting protein synthesis is no longer at the same level. However, it has been observed that macrolides in combination with another active molecule have excellent antibacterial activity. This led to the development of the idea for the synthesis of a double-acting molecule consisting of a macrolide backbone to which various other pharmacophore groups are attached. Quinolones (macrolones), acylides, nonsteroidal anti-inflammatory drugs (NSAIDs), steroids, and glycoconjugates are some of the most effective side chains attached to the macrolide ring. This final paper describes molecules found by searching the literature, which have antibacterial and/or anti-inflammatory activity. The length of the linker between the two molecules is a significant factor in the antibacterial effect. The greatest antibacterial efficacy is achieved by modification of cladinose sugar or modification in the C-3 position of the macrocyclic ring. Macrolide and steroid/NSAID hybrids showed improved activity with more specific activity due to improved accumulation within inflammatory cells. In the case of glycoconjugates, however, a reduced antibacterial effect of disaccharides in relation to monosaccharides has been confirmed, with supposedly playing steric disturbances a major role
The importance of monitoring the vitamin content of whole-day meals in kindergartens
No full textPrehrana je jedan od najvažnijih faktora razvitka i razvoja ljudskog života te očuvanja zdravlja. Pravilna prehrana djece predškolske dobi važna je zbog ubrzanog rasta i razvoja i treba se bazirati na piramidi pravilne prehrane. Podrazumijeva hranjive tvari, zastupljene i konzumirane u uravnoteženom omjeru. Vitamini su tvari organskog podrijetla koje organizmu trebaju u relativno malim količinama za rast, održavanje zdravlja i metaboličkog integriteta. Unose se prehranom ili dodacima prehrani zbog nedovoljne sinteze u ljudskom organizmu. Poznato je 13 vitamina koji se prema topljivosti dijele na vitamine topljive mastima (vitamin A, D, E i K) i one topljive u vodi (vitamini B skupine i vitamin C). Zadovoljavanje preporučenih dnevnih doza je nužno jer deficit bilo kojeg od vitamina u organizmu, uzrokuje sindrom sa tipičnim znakovima i simptomima. Cilj rada bio je izmjeriti vrijednosti vitamina A, B1, B2, B6 i vitamina C iz cjelodnevnih uzoraka hrane uzorkovanih u dječjim vrtićima te ih usporediti sa preporučenim dnevnim dozama za djecu vrtićke dobi. Prema sklopljenim ugovorima, uzorkovanje cjelodnevnih obroka hrane vrši se četiri puta godišnje, metodom slučajnih uzoraka. Uzorci se homogeniziraju i konzerviraju do provođenja saponifikacije, derivatizacije i konačnog određivanja metodom tekućinske kromatografije visoke djelotvornosti (HPLC). Prosječni dnevni unosi vitamina A, B1, B2 i C nisu, u periodu od 10 godina promatranja, svake godine zadovoljili 80% preporuka Prehrambenog standarda za planiranje prehrane u dječjim vrtićima, dok je vitamin B6 kontinuirano iznad preporuka. Praćenje sadržaja vitamina u cjelodnevnim obrocima djece važno je zbog pravovremene reakcije na eventualne nedostatke i neadekvatne unose ovih esencijalnih mikronutrijenata kod djece. Prilikom planiranja jelovnika naglasak treba biti na svježem, sezonskom voću i povrću koje ne zahtjeva daljnju termičku obradu i prirodno je bogato mikronutrijentima. Ključna je edukacija roditelja i stručnjaka koji rade s djecom o zdravoj i pravilnoj prehrani te benefitima zdravih navika stečenih u djetinjstvu.Nutrition is one of the most important factors for the growth and development of the human life and for health maintenance. Proper nutrition of preschool children is important because of their accelerated growth and development and should be based on a nutritional pyramid. It implies nutrients, represented and consumed in a balanced ratio. Vitamins are substances of organic origin that the body needs in relatively small quantities to grow, to maintain health and its metabolic integrity. They are ingested through diet or dietary supplements in cases of insufficient synthesis in the human body. There are 13 known vitamins that are divided according to their solubility into fat-soluble vitamins (vitamins A, D, E and K) and water-soluble ones (B vitamins and vitamin C). Recommended daily intake doses are necessary to be met because a deficiency of any of the aforementioned vitamins in the body causes a syndrome with typical signs and symptoms. The aim of the study was to measure the values of vitamins A, B1, B2, B6 and vitamin C from whole-day meals sampled in kindergartens and compare them with the recommendations for preschool age children. According to the concluded contracts, sampling of whole-day meals is done four times a year, by method of random samples. The samples are homogenized and preserved until saponification, derivatization and final determination by high performance liquid chromatography (HPLC). Average daily intakes of vitamins A, B1, B2 and C in the period of 10 years of observation did not meet 80% of the recommendations each year, while vitamin B6 is continuously above the recommendations of the Food Standard for diet planning in kindergartens. Monitoring the vitamin content in children's whole-day meals is important because of the timely response to possible deficiencies and inadequate intake. When planning the menu, focus should be on fresh, seasonal fruits and vegetables that do not require further heat treatment and are naturally rich in micronutrients. It is important to educate parents and kindergarten professionals about healthy and proper nutrition and the benefits of healthy habits acquired in childhood
Role of circadian genes in the chemoresistance of colon cancer cells with BRAF V600E mutation
No full textKarcinom debelog crijeva se smatra jednim od češćih karcinoma u svijetu te otprilike 5-10% svih slučajeva karcinoma debelog crijeva otpada na one sa BRAFV600E mutacijom. Unatoč sve boljim kemoterapijskim opcijama, nije primijećeno značajno poboljšanje u ishodu liječenja oboljelih sa BRAFV600E mutacijom, uglavnom zbog razvoja kemorezistencije. Cirkadijalni sustav sudjeluje u brojnim staničnim procesima poput staničnog ciklusa, popravka DNA, metabolizma i regulaciji apoptoze te je zadnjih godina porastao interes za istraživanjem mogućih poveznica između cirkadijalnog sustava i tumorigeneze. S ciljem boljeg razumijevanja mehanizama koji pogoduju razvoju kemorezistencije, odlučili smo u ovom diplomskom radu istražiti vezu između cirkadijalnih satnih gena i stečene rezistencije na BRAF V600E inhibitor vemurafenib u stanicama raka debelog crijeva sa BRAFV600E mutacijom. U tu svrhu su analizirani ekspresijski profili cirkadijalnih satnih gena u kemosenzibilnim i kemorezistentnim RKO stanicama raka debelog crijeva sa BRAFV600E mutacijom pomoću RT-PCR metode. Dobiveni rezultati ukazuju na povećanu ekspresiju PER gena u kemorezistentnim stanicama sa statističkom značajnošću p<0.05. Najveća ekspresija je zabilježena kod PER3 gena što ukazuje na moguću ulogu PER3 u razvoju rezistencije stanica raka debelog crijeva sa BRAFV600E mutacijom na vemurafenib
Mjerenje stvaranja singletnog kisika upotrebom fotodegradacijske metode
No full textFotodinamička terapija (PDT) suvremen je pristup liječenju tumora koji se oslanja na interakciju svjetlosti određene valne duljine s molekulom fotosenzibilizatora koja iz svog pobuđenog tripletnog stanja pobuđuje molekularni kisik na više energetsko stanje. U tom procesu nastaje visoko reaktivni singletni kisik. Terapijski efekt postiže se lokaliziranim citotoksičnim djelovanjem singletnog kisika na tumorsko tkivo. ROS-ovi poput singletnog kisika razaraju proteine, lipide, nukleinske kiseline i ostale stanične komponente na mjestu djelovanja. Učinkovitost fotodinamičke terapije nastoji se poboljšati modifikacijom svojstava fotosenzibilizatora. Porfirini su zbog svojeg karakterističnog apsorpcijskog i emisijskog spektra posebice zanimljivi kandidati za fotosenzibilizatore. Odabir porfirina koji su potencijalni kandidati za PDT zahtijeva razvitak pouzdanih metoda mjerenja produkcije singletnog kisika.
U ovom je radu provedeno indirektno mjerenje produkcije singletnog kisika dvaju N-metiliranih i N-oksidiranih porfirina u četiri različita otapala korištenjem fotodegradacijske metode. Fotodegradacijska metoda mjerenja produkcije singletnog kisika oslanja se na mjerenje pada fluorescencije kontrolne boje, u ovom slučaju 1,3-difenilizobenzofurana, u prisutnosti singletnog kisika. Prednost ove metode jest njena praktičnost i komercijalna dostupnost, ali treba imati na umu da rezultat ovisi o uvjetima prilikom mjerenja. Uspoređena je topivost porfirina u otapalima s obzirom na N-supstituent, te je promatran utjecaj topivosti i lipofilnosti N-supstituiranih porfirina na produkciju singletnog kisika. Utvrđeno je da svi ispitivani porfirini imaju značajan kvantni prinos singletnog kisika barem u nekima od ispitivanih otapala, te da produkcija singletnog kisika uvelike ovisi o topivosti porfirina u određenom otapalu.Photodynamic therapy (PDT) is a contemporary approach to cancer treatment that relies on interaction of light of a specified wavelength with a photosensitizer molecule that has the ability to excite molecular oxygen to a higher energy state. During that process, molecular oxygen receives energy from the photosensitizer in its excited triplet state, thus becoming highly reactive singlet oxygen. The therapeutic effect is achieved by cytotoxic activity of singlet oxygen targeted at tumor tissue. Reactive oxygen species such as singlet oxygen are capable of damaging proteins, lipids, nucleic acids and other cellular componets at the site of impact. By modifying the properties of the photosensitizer one can improve the effectiveness of PDT. Due to ther characteristic absorption and emission spectra, porphyrins are particularly interesting candidates for usage as photosensitizer molecules. Choosing a porphyrin that is a potential candidate for PDT requires development of reliable methods for measuring singlet oxygen production.
In this work, singlet oxygen production by two N-methylated and two N-oxidated porphyrins in four solvents was measured indirectly using photodegradation method. The aforementioned method relies on measuring the decrease of fluorescence emission by control dye, in this case DPBF, in the presence of singlet oxygen. The advantage of this method is simple application and commercial availability, although it has to be noted that the result vastly depends on conditions during experiment. Solubility of porphyrins in various solvents was compared in regards to their N-substituent as well as the effect of solubility of N-substituted porphyrins on singlet oxygen production. It was found that all of the examined porphyrins have significant singlet oxygen production at least in some of the solvents. Also, it was shown that singlet oxygen production depends on solubility of the porphyrin in a given solvent
Development of a UPLC-MS method for measurement of the chromatographic hydrophobicity index (CHI) at pH 2.7 and 10.5
No full textLipofilnost se uz konstantu ionizacije i topljivost smatra najvažnijim fizikalno-kemijskim svojstvom jer ima izravan utjecaj na svojstva apsorpcije, distribucije, metabolizma i izlučivanja (ADME) potencijalnog lijeka u organizmu, kao i na toksičnost. Predstavlja afinitet molekule ili dijela molekule za lipofilnu okolinu te se izražava preko logaritama particijskog ili distribucijskog koeficijenata (log P ili log D). Zbog značajnog utjecaja na svojstva potencijalnog lijeka određuje se već u vrlo ranim fazama istraživanja lijeka. Tradicionalne metode određivanja lipofilnosti, poput metode izmućkivanja, ograničene su na topljive spojeve te zahtijevaju veću količinu spoja i dulje vrijeme. Stoga se u ranim fazama istraživanja zamijenjuju s indirektnim visokoprotočnim analizama, kao što je obrnuto-fazna tekućinska kromatografija visoke djelotvornosti (HPLC). Jedna od takvih indirektnih metoda je metoda za određivanje kromatografskog indeksa hidrofobnosti (CHI) koja se zasniva na preračunavanju gradijentnih vremena zadržavanja analita na koloni u CHI uz pomoć jednadžbe dobivene linearnom regresijskom analizom.
Cilj ovog rada bio je razvijanje metode za određivanje CHI pri kiselim (pH 2.7) i bazičnim (pH 10.5) uvjetima pokretne faze koristeći obrnuto-faznu tekućinsku kromatografiju ultra visoke djelotvornosti (RP-UPLC) spregnutom sa spekrometrijom masa (MS). Kromatografski indeks hidrofobnosti ispitan je pri pH 2.7, 7.4 i 10.5 za set od 30 spojeva, podijeljenih u slijedeće skupine: kiseli, bazični, neutralni, amfoterni i makrolidi. Pomoću komercijalnog software-a ACD/Percepta određene su in silico vrijednosti za log D pri pH 2.7, 7.4 i 10.5.
Metoda određivanja CHI vrijednosti pri pH 2.7 i pH 10.5 uspješno je uvedena na RP-UPLC/MS sustavu te je pokazala visoku preciznost. Istovremeno je prikazan i utjecaj starenja kromatografske kolone na CHI vrijednosti. Određivanjem CHI vrijednosti pri tri različita pH određena su kiselo-bazna svojstva za sve ispitane spojeve te su dobivene visoke korelacije sa in silico izračunatim vrijednostima za većinu spojeva.Lipophilicity is considered to be the most important physicochemical property, in addition to the ionization constant and solubility, as it has a direct impact on the absorption, distribution, metabolism and excretion (ADME) properties of a potential drug in the body, as well as on toxicity. It represents the affinity of a molecule or part of a molecule for a lipophilic environment and expresses the logarithm of the partition or distribution coefficient (log P or log D). Due to the significant impact on the properties of the potential drug, it is usually determined already in the very early stages of drug discovery. Traditional methods of determining lipophilicity, such as the shake-flask method, are limited to soluble compounds and require a larger amount of compound and a longer time. Therefore, they are being replaced by indirect high-throughput analyses, such as reversed-phase high-performance liquid chromatography (HPLC) in the early stages of discovery. Such an indirect measurement is the method for determining the chromatographic hydrophobicity index (CHI), which is based on calculating CHI values from gradient retention times of the analyte using the equation obtained by linear regression analysis.
The aim of this work was to develop a method for determining CHI under acidic (pH 2.7) and basic (pH 10.5) conditions of the mobile phase using reversed phase ultra high performance liquid chromatography (RP-UPLC) coupled to mass spectrometry (MS). CHI was determined at pH 2.7, 7.4 and 10.5 for a set of 30 compounds, divided into the following groups: acidic, basic, neutral, amphoteric and macrolides. In silico values for log D at pH 2.7, 7.4 and 10.5 were calculated using a commercial software ACD/Percepta.
A method with high precision for determining CHI at pH 2.7 and pH 10.5 was successfully implemented using RP-UPLC/MS. The influence of an aging chromatographic column in the method was also shown. By determining of CHI values at three different pH values it was possible to characterize the acid-base character for all tested compounds. A good correlation between measured CHI values and with in silico predictions was obtained for most compounds
Tripyridylporphyrins in photodynamic inactivation of legionellae
No full textGram-negativna bakterija Legionella pneumophila je sveprisutna u vodenim i vlažnim područjima. Zbog njene rezistentnosti na određene skupine protumikrobnih lijekova, nužni su novi pristupi u eradiciranju ove bakterije. U ovom radu istražila se mogućnost primjene fotodinamičke inaktivacije u dezinfekciji vode koja bi mogla imati primjenu u budućnosti kako bi se onemogućilo širenje zaraze uzrokovane navedenom bakterijom. Fotodinamička inaktivacija je novi pristup u eradikaciji bakterija te zahtijeva prisustvo fotosenzibilizatora, molekularnog kisika te svjetlosti. Izlaganje molekule fotosenzibilizatora svjetlosti uzrokuje nastanak reaktivnih kisikovih vrsta ili singletnog kisika koji uzrokuje smrt bakterijske stanice. Kao fotosenzibilizator u ovom radu se koristio amfifilni porfirin, 5-(4-oktadec-9-enamidofenil)-10,15,20-tris(3-piridil)porfirin. Određena je minimalna efektivna koncentracija dilucijskom metodom koja se koristila u testu fotoinaktivacije. Svi eksperimenti provedeni su u sterilnoj vodi iz slavine te se koristila ljubičasta svjetlost intenziteta 40 mW/cm2. U istraživanju se pokazalo kako je ispitivani spoj djelotvorniji nakon svjetlosne aktivacije te da ima učinak protiv stvaranja biofilma. Također se provelo ispitivanje djelovanja otopine spoja 2,5 mjeseca nakon pripreme.
U istraživanju su određene vrijednosti koncentracija MEKs = 0,391 μM, MEK2,5 = 0,195 μM. Nakon izvođenja testa toksičnosti u mraku određene su vrijednosti MEKs = 6,25 μM te MEK2,5 = 25 μM. Koncentracije koje inhibiraju adheziju iznosile su MEK = 0,391 μM nakon izlaganja svjetlosti te MEK = 0,781 μM bez izlaganja svjetlosti.Gram-negative bacterium Legionella pneumophila is ubiquitous in water and moist areas. Because of its resistance to certain types of antimicrobial drugs, the new ways of eradication are needed. The possibility of application of photodynamic inactivation in water disinfection, which may have a potential application to arrest the spreading of infection caused by aforementioned bacterium, will be presented in this work. Photodynamic inactivation is a new approach in eradication of bacteria and requires the presence of photosensitizer, molecular oxygen and light. Exposure of photosensitizer to light results in generation of reactive oxygen species or singlet oxygen which cause the death of bacterial cell. Amphiphilic porphyrin, 5-(4-octadec-9-enamidophenyl)-10,15,20-tris(3-pyridyl)porphyrin, was used as a photosensitizer in this work. Minimal effective concentration has been determined by microdillution method and it was used in the photoinactivation test. All experiments were conducted in tap water and violet light with 40 mW/cm2 fluenece was used. The tested compound was shown to be more effective after the light activation and also has an effect against the production of biofilms. An examination of the effect of the solution 2,5 months after the preparation was also conducted.
Concentrations which are determined in this research include MECs = 0,391 μM, MEC2,5 = 0,195 μM. After the dark toxicity test concentrations valued MECs = 6,25 μM and MEC2,5 = 25 μM. Concentrations which inhibits adhesion valued MEC = 0,391 μM after exposure to light and MEC = 0,781 μM after dark toxicity test