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Synthesis of new tripyridyl amphiphilic porphyrins for use in photodynamic therapy
No full textFotodinamička terapija je selektivna metoda liječenja tumora i ostalih bolesti koja kombinira elektromagnetsko zračenje određene valne duljine i fotosenzibilizator, tvar osjetljivu na navedeno zračenje, uz prisutnostost kisika kako bi uništila ciljane stanice bez nepovoljnih sistemskih učinaka na organizam pacijenta. Dobar fotosenzibilizator uz niz fotofizičkih treba imati i povoljan omjer hidrofilnih i lipofilnih svojstava kako bi se mogao administrirati u vodenom mediju i ne stvarati agregate, a istovremeno prelaziti staničnu membranu i nakupljati se u tumorskim stanicama. Poznato je kako porfirini imaju većinu poželjnih svojstava dobrih fotosenzibilizatora, a studije ukazuju kako trikationski porfirini s 3-piridilnim supstituentima uz jednu slobodnu meso poziciju za daljnju funkcionalizaciju čine dobar temelj strukture fotosenzibilizatora. Također, konjugacija s masnim kiselinama može povećati fotodinamičku aktivnost i selektivnost za tumorske stanice.
Kako bi nastavili istraživanje u navedenom pravcu u ovom su radu sintetizirani novi 3- i 4-piridilni derivati porfirina. Konjugati su pripremljeni pomoću asimetričnog tripiridilnog porfirina dobivenog modificiranom Adler - Longo sintezom i njegovim daljnjim modifikacijama; konjugacijom sa stearoil- i oleoil-kloridom te kvaternizacijom piridinskih dušika jodmetanom i meta-klorperbenzojevom kiselinom. Tako su pripravljeni amfifilni kationski i zwitterionski spojevi s dugim alifatskim lancima koji bi mogli imati odgovarajuću ravnotežu hidrofilnih i lipofilnih svojstava u molekuli za potencijalnu primjenu u fotodinamičkoj terapiji.
Svi novo pripravljeni spojevi izolirani su u čistom stanju, a strukture su potvrđene i okarakterizirane 1H i 13C NMR, UV/VIS, fluorescentnim i masenim spektrima. Spojevi su namijenjeni daljnjem testiranju in vitro fotodinamičke aktivnosti u bakterijskim i tumorskim stanicama.Photodynamic therapy is a selective method for treating cancer and various other diseases which combines light of appropriate wavelength and photosensitizer sensitive to that light with oxygen to produce toxic effects on targeted cells in immediate surroundings without systemic adverse effects on a patient. Ideal photosensitizer, apart certain photophysical properties, also needs to have appropriate ratio between hydrophilic and lipophilic properties in order to be administered in aqueous medium without aggregating, to traverse cell membranes through lipid layers, and accumulate in tumor cells. Porphyrins are known to possess many of the desired properties of a good photosensitizer and studies have shown that tricationic porphyrins with 3-pyridyl substituents with one free meso position for further functionalization make a good photosensitizer basis. Moreover, conjugation with fatty acids has shown to increase compound's photodynamic activity and selectivity for tumor cells.
In order to further investigate the effects of different modifications, in this research new 3- and 4-pyridyl derivatives of porphyrins were synthesized. Conjugates were prepared from asymmetric tripyridyl porphyrins made in modified Adler – Longo synthesis and their further modifications; bonding with long alkyl chains of stearic and oleic acid in reactions with their chlorides and quaternization of pyridyl nitrogens with iodomethane and meta-chloroperbenzoic acid. Thus, final amphiphilic cationic and zwitterionic compounds with long alkyl chains are prepared that might have the required balance between hydrophilic and lipophilic properties for potential use in photodynamic therapy.
All new compounds were isolated pure and their structures were confirmed and analyzed by 1H and 13C NMR, UV/VIS, fluorescence spectroscopy and mass spectrometry. Future work involves testing these products for in vitro photodynamic activity in bacterial and tumor cells
SYNTHESIS, CHARACTERIZATION AND SELF-ASSEMBLY OF SMALL PEPTIDIC GELATORS BASED ON AMYLOID β- PROTEIN
Get PDFKlasičnim sintetskim metodama peptidne kemije u otopini sintetizirana je serija novih
peptidomimetika, tripeptidnih derivata koji u svojoj strukturi sadrţe različite kombinacije
aminokiselinskih slijedova (FFA, FAF, AFF) i s različitim zaštitnim grupama na N i C
terminalnim završecima. Ispitana su gelirajuća svojstva pripravljenih spojeva u vodi,
organskim otapalima i smjesama različitih otapala. Acetilni tripeptidi s aminokiselinskim
slijedom FFA (Phe-Phe-Ala) pokazali su se kao dobri gelatori vode i polarnih otapala (6), ali i
aromatskih otapala (5). Acetilni derivati s ostalim aminokiselinskim slijedovima (FAF i AFF)
pokazali su izrazito slaba gelirajuća svojstva kao i butirilni tripeptidi sa slijedom FFA.
Tripeptidi s benziloksikarbonilom (Z) na N terminalnoj strani (28, 32 i 37) su jako efikasni
gelatori aromatskih otapala (o-, m- i p-ksilena i tetralina) te dekalina. Morfologija gelskih niti
odreĎena je transmisijskom elektronskom mikroskopijom (TEM). Samoorganizacija molekula
i supramolekularne interakcije u novim tripeptidnim gelovima proučavane su različitim
spektroskopskim metodama (NMR, FTIR, CD) koje su ukazale na postojanje
samoudruţivanja molekula, strukture β-nabrane ploče (paralelno ili antiparalelno orijentirane)
povezane vodikovim vezama kod 5 i 6 te kod Z-zaštićenih tripeptida (28, 32 i 37). UV-Vis i
fluorimetrijskom spektroskopijom te laserskim pretraţnim konfokalnim mikroskopom
ispitano je vezanje tripeptida 6 na amiloidne boje (tioflavin T i kongo-crvenilo) u fiziološkim
uvjetima. Fluorescencijskom titracijom vodene otopine tripeptida 6 s tioflavinom T dolazi do
porasta emisije te boje i formiranja kompleksa stehiometrije 1:1 s konstantom stabilnosti log
K = 2,48. Kongo-crvenilo s tripeptidom 6 tvori proziran gel u vodi dok sam spoj 6 stvara
mutan hidrogel pri čemu dolazi do značajnijeg porasta emisije fluorescencije spoja kongocrvenilo
u gelu u odnosu na otopinu gdje je neznatan porast emisije. Hidrogel kongo-crvenila
i tripeptida 6 čine tanke niti promjera 10-15 nm dok TEM samog hidrogela 6 prikazuje
prisutnost ravnih traka promjera 50-500 nm što ukazuje na promjenu morfologije gelova.
Laserskim pretraţnim konfokalnim mikroskopom pokazano je da se obje amiloidne boje veţu
na gelske niti tripeptida 6. Hidrogel tripeptida 6 omogućio je u fiziološkim uvjetima
preţivljenje i proliferaciju stanica HEK293T in vitro te se pokazao kao potencijalni
biomaterijal za primjenu u tkivnom inţenjerstvu. Dosadašnja istraţivanja su pokazala da bi
novosintetizirani hidrogelator 6 mogao posluţiti kao potencijalni minimalistički model
agregiranog Aβ-proteina i omogućiti precizan dizajn i razvoj novih efikasnijih molekula
inhibitora ili detektora agregiranjaSeries of tripeptide FFA, FAF and AFF derivatives with different protecting groups was
prepared using classical methods of solution-state peptide synthesis. Prepared tripeptides were
tested for gelation of water, various organic solvents and mixtures of solvents. Acetyl FFA
derivatives exhibited gelation of water, polar solvents (6) and aromatic solvents (5). However,
acetyl FAF and AFF derivatives and butyryl FFA derivatives showed poor gelation abilities.
Tripeptides with benzyloxicarbonyl protecting group (28, 32 and 37) exhibited strong gelation
of aromatic solvents (o-, m- and p-xylene and tetraline) and decaline. Morphology of prepared
gels was investigated by Transmission Electron Microscopy (TEM). Organisation in gel
assemblies at the molecular and the supramolecular level determined by using spectroscopic
methods (NMR, FTIR, CD) pointed towards the β-sheet type of hydrogen bonding selfassociation
of tripeptides in gel aggregates. Binding studies of tripeptide 6 with amyloid dyes,
Congo Red and Thioflavin T (ThT) were carried out using fluorescence spectroscopy and
confocal microscopy. Fluorescence titration of tripeptide 6 aqueous solution bellow its
minimal gelation concentration with Thioflavin T showed increase of ThT emission with
increased tripeptide concentration and formation of the 1:1 complex with the association
constant, log K = 2,48. Congo Red forms transparent hydrogel with a tripeptide 6 and shows
increased fluorescence emission compared to aqueous solution of Congo Red. TEM of the
tripeptide hydrogel with Congo Red revealed a change in fiber morphology compared to 6
hydrogel. TEM images of the nanofibrous hydrogel network show the presence of a mixture
of fibers and straight ribbons with diameters in the range of 50–500 nm while the hydrogel
network together with Congo Red contains small fibrils with diameters in range of 10-15 nm.
Confocal microscopy revealed that the both dyes bind the hydrogel fibers. As a potential
biomaterial, 6 was established as a stable and biocompatible physical support for HEK293T
cells in vitro. Tripeptide 6 efficiently supported survival and promoted proliferation of
HEK293T cells encapsulated within a three-dimensional nanofiber network. Furthermore,
these results suggest the need for further evaluation of in vitro biocompatibility and
bioactivity of this tripeptide on neural stem cells upon encapsulation for possible tissue
engineering application. Likewise, tripeptide 6 could serve as minimalistic model of
aggregated Aβ-protein and enable development of new amyloid aggregation inhibitors
Ispitivanje fotodinamičkog učinka amfipatskog porfirina na legionele
No full textLegionella pneumophila je Gram-negativna bakterija čije je prirodno stanište voda, uključujući
rijeke, jezera i potoke. L. pneumophila je uzročnik pontiačke groznice i legionarske bolesti, a
distribucijski sustavi za vodu, rashladni tornjevi i ovlaživači zraka samo su neki od vodenih
izvora infekcije. Unatoč provođenju protuepidemijskih mjera sve češća je pojava legioneloza
stoga je potrebno razviti nove metode za uklanjanje legionele iz vode. Jedan od pristupa je
protumikrobna fotodinamička terapija koja uključuje zajedničko djelovanje fotosenzibilizatora,
molekularnog kisika i vidljivog svjetla određene valne duljine u svrhu produkcije singletnog
kisika i kisikovih reaktivnih vrsta koji ubijaju stanicu. U radu je uspoređeno djelovanje
novosintetiziranog amfipatskog porfirina s dva hidrofilna porfirina za evaluaciju moguće
fotodinamičke inaktivacije bakterije u vodi. Određivane su minimalne inhibitorne i minimalne
baktericidne koncentracije u bujonu i minimalne efektivne koncentracije u vodi dilucijskom
metodom. Korišteno je crveno svjetlo ukupne doze svjetla 7,2 J cm-2 ili 24 J cm-2 te ljubičasto
svjetlo ukupne doze svjetla 12 J cm-2. Najdjelotvornijim se pokazao amfipatski porfirin dok se
aktivnost hidrofilnih porfirina minimalno razlikuje u korist tetrakationskog. Duže osvjetljavanje
crvenim svjetlom slabijeg protoka i manje ukupne doze svjetla se pokazalo više učinkovitim od
kraćeg osvjetljavanja crvenim svjetlom jačeg protoka i veće ukupne doze svjetla vjerojatno
zbog dužeg izlaganja bakterija svjetlu. Spojevi u sterilnoj vodi su iskazali antimikrobno
djelovanje i bez djelovanja svjetla, ali se toksičnost poveća nakon aktivacije svjetlom.Legionella pneumophila is a Gram-negative bacteria whose natural habitat is water, including
rivers, lakes and streams. L. pneumophila causes Pontiac fever and Legionnaires' disease
with some of the water sources of infection being water distribution systems, cooling towers
and humidifiers. Despite anti-epidemic measures, legionellosis outbursts still occur very
frequently and because of that it is necessary to develop new methods to remove Legionella
from the water. One of the aproaches is antimicrobial photodynamic therapy, which includes
combined activity of photosensitizer, molecular oxygen and visible light of appropriate
wavelength to create singlet oxygen and oxygen reactive species leading to cell death. In this
study we compared the activity of the newly synthesized amphipathic porphyrin with two
hydrophilic porphyrins for possible photodynamic inactivation of bacteria in the tap water. The
results were derived from the determination of minimum inhibitory and minimum bactericidal
concentration in broth and minimum effective concentration in tap water by the microdilution
method. A red light with the total dose of light 7,2 J cm-2 or 24 J cm-2 was used and violet light
with total dose of light 12 J cm-2. The most efficient compound was amphiphilic porphyrin while
the activities of the hydrophilic porphyrins differ minimaly but in favor of the tetracationic
porphyrin. Longer illumination with lower dosage of red light was shown to be more effective
than shorter illumination with red light with higher fluence rate and higher total dose of light
probably due to the longer exposure of bacteria to the light. In the sterile water, compounds
showed antimicrobial activity without illumination, but the toxicity increases after light
activation
Endokanabinoidni sustav – njegova uloga u zdravlju i bolesti
No full textEndokanabinoidni sustav skup je enzima, receptora i lipidnih molekula koji igraju
važnu ulogu u fiziologiji čovjeka. Distribuiran u mozgu i na periferiji, ovaj sustav sudjeluje u
održavanju energetske homeostaze poticanjem apetita i sprječavanjem povraćanja, smanjenju
širenja upale nakon ozlijede tkiva te općoj kvaliteti života ublažavanjem boli i grčenja mišića
kod teških patofizioloških stanja. Navedeni učinci samo su neke od uloga koje imaju
kanabinoidi u ljudskom organizmu, a iscrpna istraživanja provode se u svrhu legalizacije
njihove kliničke primjene.
Trenutno dostupni lijekovi poznatog omjera fitokanabinoida poput oromukoznog
spreja Sativex® primjenjuju se prvenstveno za olakšavanje mišićnog spazma i urgentne
inkontinencije kod multiple skleroze, a preostala dva (Marinol® i Cesamet®) dolaze u formi
kapsula i propisuju se u SAD-u, Kanadi, Novom Zelandu i diljem Europe zbog antiemetičkih
svojstava (karcinom) te kao stimulatori apetita (AIDS). U Republici Hrvatskoj od 15.
listopada 2015. godine legalizirana je primjena medicinskog kanabisa. Potencijal u narednim
godinama leži u povećavanju tonusa endogenih ekvivalenata tetrahidrokanabinola (THC-a)
inhibicijom njihovih hidrolaza, te CB2 selektivnih liganada koji nemaju psihotropne učinke.
Cilj ovog rada bio je obraditi pozitivne i negativne učinke ove skupine molekula na
zdravlje čovjeka. Značajno antikancerogeno djelovanje zabilježeno je apliciranjem agonista
kanabinoidnih receptora u in vitro i in vivo modelima najtežeg oblika tumora mozga. Osim
inhibicije angiogeneze, agonisti CB1 i CB2 receptora spriječavaju rast tumorskog tkiva
stimulacijom de novo sinteze ceramida i aktivacijom stresnog odgovora endoplazmatskog
retikuluma koji uzrokuje autofagijom-posredovanu smrt tumorskih stanica. S palijativnog
aspekta, nedavno je otkriven antinociceptivni mehanizam kojim ova skupina molekula djeluje
na područje mozga zaduženo za procesiranje boli supresijom GABAergičke transmisije.
Ovakav pristup daje nadu za razvoj novih terapeutskih pristupa za smanjenje boli kod bolesti
poput reumatoidnog artritisa, dijabetičke polineuropatije, fibromialgije i čak epilepsije. Pritom
valja imati na umu da najpotentniji kanabinoidni pripravci sadrže psihoaktivni THC koji
usljed dugoročne izloženosti vodi ka ovisnosti, depresiji i gubitku pamćenja, a pokazao se
rizičnim faktorom u razvoju mentalnih bolesti. Osim toga, ovaj članak stavlja naglasak na
negativan učinak THC-a na fertilitet muškaraca, proces spermatogeneze i funkciju spermija
usljed promjene u ravnoteži endokanabinoida spolnog sustava.The endokanabinoid system combines a group of enzymes, receptors and lipid
molecules which play an important role in human physiology. Distributed in the brain and in
the periphery,this system participates in maintaining energy homeostasis by stimulating
appetite and preventing vomiting, reducing inflammation after tissue injury and raising
overall quality of life by alleviating pain and muscle cramps in severe pathophysiological
conditions. The aforementioned effects are small part of the potency of cannabinoids, and
extensive research is carried out in order to legalize their clinical application.
Currently available drugs with known phytokanabinoid ratio such as Sativex® are
primarily used to relieve muscular spasms and urgent incontinence in multiple sclerosis, while
the remaining two (Marinol® and Cesamet®) are prescribed in the United States, Canada,
New Zealand and throughout Europe due to antiemetic properties (carcinoma) and as an
appetite stimulator (AIDS). In the Republic of Croatia since October 15th 2015, the use of
medical cannabis is legalized. Potential in the coming years lies in increasing tone of
endogenous THC equivalents by inhibiting their hydrolysis, and development of CB2
selective ligands which do not exhibit psychotropic effects.
The aim of this paper was to address the positive and negative effects of this group of
molecules on human health. Significant anticancer activity was observed by applying
cannabinoid receptor agonists in in vitro and in vivo models of the most malignant brain
tumor. In addition to inhibition of angiogenesis, CB1 and CB2 receptor agonists inhibited
tumor tissue growth by stimulating de novo synthesis of ceramide and activating ER stress
response that causes autophagy-mediated death of tumor cells. From a palliative point of
view, an antinociceptive mechanism has recently been discovered by which this group of
molecules acts on the brain region responsible for processing pain by suppressing GABAergic
transmission. This approach gives hope in the future to reduce pain in diseases such as
rheumatoid arthritis, diabetic polyneuropathy, fibromyalgia and even epilepsy. It should also
be considered that the most potent cannabinoid-based drugs contain psychoactive THC which,
after long-term exposure, leads to addiction, depression and memory loss, and also has shown
to be a risk factor for development of mental illnesses. Additionally, this article highlights the
negative impact on male fertility, spermatogenesis, and sperm function of THC due to
changes in the balance of endocannabinoids located in the male reproductive system
An unpredicted aggregation-critical region of the actinpolymerizing protein TRIOBP-1/Tara, determined by elucidation of its domain structure
Get PDFAggregation of specific proteins in the brains of patients with chronic mental illness as a result of disruptions in proteostasis is an emerging theme in the study of schizophrenia in particular. Proteins including DISC1 (disrupted in schizophrenia 1) and dysbindin-1B are found in insoluble forms within brain homogenates from such patients. We recently identified TRIOBP-1 (Trio-binding protein 1, also known as Tara) to be another such protein through an epitope discovery and proteomics approach by comparing post-mortem brain material from schizophrenia patients and control individuals. We hypothesized that this was likely to occur as a result of a specific subcellular process and that it, therefore, should be possible to identify a region of the TRIOBP-1 protein that is essential for its aggregation to occur. Here, we probe the domain organization of TRIOBP-1, finding it to possess two distinct coiled-coil domains: the central and C-terminal domains. The central domain inhibits the depolymerization of F-actin and is also responsible for oligomerization of TRIOBP-1. Along with an N-terminal pleckstrin homology domain, the central domain affects neurite outgrowth. In neuroblastoma cells it was found that the aggregation propensity of TRIOBP-1 arises from its central domain, with a short “linker” region narrowed to within amino acids 324–348, between its first two coiled coils, as essential for the formation of TRIOBP-1 aggregates. TRIOBP-1 aggregation, therefore, appears to occur through one or more specific cellular mechanisms, which therefore have the potential to be of physiological relevance for the biological process underlying the development of chronic mental illness
S-ADENOSYLHOMOCYSTEINE HYDROLASE DEFICIENCY: MOLECULAR MECHANISMS OF NOVEL DISORDER
Get PDFUvod i cilj
Nedostatak S-adenozilhomocistein hidrolaze (AHCY) je poremećaj uzrokovan mutacijama u genu ahcy čime je smanjena aktivnost proteina AHCY. AHCY ima ključnu ulogu u pravilnom odvijanju ciklusa aminokiseline metionina u stanici, stoga nedostatak njegove funkcije uzrokuje težak metabolički poremećaj. Kao jedini enzim koji hidrolizira SAH, snažni inhibitor staničnih metiltrasferaza, AHCY ima i indirektnu ulogu u održavanju metilacijskog statusa stanice. Klinička slika ovog potencijalno letalnog oboljenja je karakterizirana kombinacijom mišićnih, neuroloških i jetrenih poremećaja. Unatoč dokazanoj esencijalnosti AHCY, promjene u molekularnim mehanizmima stanice u stanju nedostatka AHCY su dosada nedovoljno istražene. Cilj ove doktorske disertacije je analiza i razumijevanje molekularnih i staničnih uloga AHCY s krajnjom svrhom probira potecijalnih biomarkera oboljenja.
Materijali i metode
U sklopu ove disertacije su rađene studije novootkrivene Y328D mutante potvrđene u pacijentici oboljeloj od nedostaka funkcije AHCY, a za dodatna istraživanja na raspolaganju su bili i primarni fibroblasti pacijentice. Zbog ograničenja u radu s primarnim fibroblastima, tijekom izrade doktorske disertacije su se pripremili, validirali i istraživali modelni stanični sustavi za što se koristila kombinacija metoda klasične biologije i različitih omiks i visokoprotočnih tehnika.
Rezultati
Po prvi puta je tijekom istraživanja AHCY korištena metoda kojom se na temelju fluorescencije dviju komplementiranih podjedinica proteina Venus s kojima su označeni proteini od interesa može pratiti njihova interakcija u ljudskim stanicama. Dokazano je da Y328D mutanta gubi sposobnost homodimerizacije, a metoda je također uspješno prilagođena na visokoprotočno skeniranje interaktora proteina AHCY. Nadalje, uočena je smanjena razina mutiranih AHCY mRNA i proteina u fibroblastima pacijenta i u stanicama sa stabilno eksprimiranim mutiranim AHCY. U istim stanicama je također mikroskopijom pokazano da mutacije uzrokuju povećanu ili smanjenu količinu proteina u jezgri stanica u odnosu na divlji tip. U stanicama hepatocelularnog karcinoma s utišanim AHCY smanjena je proliferacija i migracija te su aktivirani stanični putevi koji signaliziraju oštećenje DNA. Tijekom analize transkriptoma i proteoma fibroblasta pacijenta i stanica hepatocelularnog karcinoma s utišanim AHCY, uočena je moguća veza proteina AHCY s patologijom različitih neuroloških, jetrenih i mišićnih oboljenja, a predložena su i dva potencijalna biomarkera oboljenja.
Zaključci
Promjene razine AHCY mRNA i proteina, kao i lokalizacije AHCY u stanici koje su uočene u svih istraživanih mutanti proteina AHCY mogu biti odgovorne ili pridonositi patologiji kod oboljenja nedostatka AHCY. Prema predloženom mehanizmu AHCY ima utjecaj na oštećenje DNA i stanični ciklus. Dva potencijalna biomarkera su predložena s ciljem nastavka njihovog budućeg istraživanja u svrhu prognostike i dijagnostike oboljenja nedostatka AHCY.Introduction and aims
S-adenosylhomocysteine hydrolase (AHCY) deficiency is a disorder caused by lowered enzymatic activity of AHCY protein due to the mutations in ahcy gene. AHCY has a key role in proper functioning of the methionine cycle in cell, therefore the lack of AHCY function causes severe metabolic disorder. Since AHCY is a single enzyme that hydrolyses SAH, a strong inhibitor of cellular metiltransferazes, it has a central role in maintaining the methylation status of the cell. Clinical presentation of this potentially lethal disorder includes a combination of muscular, neurological and hepatic disorders. Despite the essential activity of AHCY, changes in molecular and cellular mechanisms in the state of AHCY deficiency have so far been poorly investigated. The aim of this doctoral thesis is the analysis and understanding of molecular and cellular roles of AHCY and prediction of potential disease biomarkers.
Materials and methods/methodological approach
In this thesis, we performed studies of newly discovered Y328D mutant confirmed in the recent case of AHCY deficiency. Patient fibroblasts were obtained from the mentioned case, however, due to the limitations in experimental work with primary fibroblasts, we prepared, validated and investigated model cellular systems by combining methods ranging from classical biology approach to different omics and high throughput techniques.
Results
A method that enables visualization of protein interactions in human cells based on fluorescence of two complemented parts of Venus protein fused to proteins of interest was used for the first time to research AHCY protein. It is proven that Y328D mutant lacks the ability to form homodimes in human cells, and the method has been also successfully adapted to high throughput screening of AHCY protein interactors. Further, we report lower levels of mutant AHCY mRNA and protein in patient fibroblasts as well as cells that stably express AHCY mutant proteins. Microscopy revealed higher or lower amounts of mutant AHCY protein in cell nuclei in mentioned cells when compared to wild type. In hepatocellular carcinoma cells with a silenced AHCY expression we report lowered proliferation and migration as well as activation of DNA damage induced cellular pathways. While analyzing the transcriptome and proteome of patient fibroblasts and hepatocellular carcinoma cells with silenced AHCY expression, potential implications of AHCY protein in the pathology of various neurological, hepatic and muscular diseases are recognized, and two potential disease biomarkers are suggested.
Conclusions
Changes in levels of mutant AHCY mRNA and protein, as well as changed cellular localization of mutant AHCY could be responsible for or contribute to the pathology of the AHCY deficiency. As proposed by our mechanism, AHCY can imapact cellular DNA damage and cell cycle. Two biomarkers are suggested for further research and usage in prognostics and diagnostics of AHCY deficiency
Konjugati porfirina u fotodinamičkoj terapiji
No full textKarcinom je i dalje vodeći uzrok smrti na globalnoj razini, bez obzira na napredak
u liječenju i detekciji istih. Konvencionalni kemoterapeutski agensi korišteni u liječenju
tumora su nespecifične toksičnosti te osim tumorskih, napadaju i zdrave stanice što dovodi
do raznih nuspojava. Nadalje, tumorske stanice mogu nakon nekog vremena razviti
rezistenciju na kemoterapeutike. Stoga, potreba za novim protutumorskim pristupima
svakodnevno raste. Jedan od obećavajućih pristupa je fotodinamička terapija. Ova terapija
kombinira zasebno tri netoksične komponente: kisik, fotosenzibilizator i svjetlost.
Ozračivanjem fotosenzibilizatora svjetlošću odgovarajuće valne duljine u blizini
molekularnog kisika dolazi do stvaranja visoko reaktivnog i toksičnog singlet kisika. Potonji
može uzrokovati oksidativno oštećenje tkiva u kojem se nalazi. Stoga je vrlo bitno da se
fotosenzibilizator lokalizira isključivo u tumorsko tkivo kako ne bi došlo do oštećenje
zdravog tkiva. U tom pogledu, kao najučinkovitiji spojevi su se pokazali upravo porfirini.
Veliki utjecaj na specifičnost porfirina ima njegova topljivost, tumorska distribucija i
mogućnost prolaska kroz tumorsku membranu. Navedena specifičnost se pokušavala
povećati na brojne načine, no kao najefikasniji način se pokazala konjugacija porfirina s
određenim biomolekulama. Zbog lake dostupnosti i jednostavnog modificiranja, peptidi su
se pokazali kao izvrsni vektori za konjugaciju. U ovom radu je prikazana konjugacija
porfirina s antimikrobnim peptidom nizinom. Očekuje se da će amfipatska svojstva
konjugata i prisutnost nizina u strukturi pospješiti topljivost fotosenzibilizatora i njegovo
prodiranje kroz tumorsku membranu. Navedeno pozitivno utječe na specifičnost i
efikasnost fotosenzibilizatora, a time i same fotodinamičke terapije.Carcinoma is still the leading cause of death globally, regardless of progress in their
treatments and detection. Conventional chemotherapeutic agents used in the treatment of
tumors have non-specific toxicity. Apart from tumor, they are attacking healthy cells as
well, which leads to various side effects. Furthermore, tumor cells may eventually develop
resistance to chemotherapy. Therefore, the need for new antitumor approaches is
increasing every day. One of the promising approaches is photodynamic therapy. This
therapy combines separately three nontoxic components: oxygen, photosensitizer and
light. After the photosensitizer is irradiated with the light of the appropriate wavelength in
the presence of the molecular oxygen, a highly reactive and toxic singlet oxygen is created.
The latter can cause oxidative damage to the tissue in which it is present. Therefore, it is
very important that the photosensitizer is localized solely in the tumor tissue so as to avoid
damaging the healthy tissue. In this respect, so far the most effective compounds were
porphyrins. Solubility, distribution within tumor and the ability to pass through the tumor
membrane greatly influence the specificity of a porphyrin. There were different efforts to
increase mentioned specificity, but the most efficient way was shown to be conjugation of
porphyrin with certain biomolecules. Peptides are easily accessible and can be simply
modified, and because of that they are proved to be excellent conjugation vectors. In this
work, the conjugation of porphyrin with antimicrobial peptide nisin is shown. Amphiphilic
properties of the conjugate and the presence of nisin in its structure are expected to
enhance solubility of the photosensitizer and its penetration through the tumor membrane.
This can positively affect the specificity and efficiency of the photosensitizer, thus
photodynamic therapy itself
Preparation and characterization of nanolipid formulations of Ambrosia Elatior proteins
No full textAlergijske reakcije (reakcije preosjetljivosti) predstavljaju značajan medicinski
problem modernog društva i načina života. Posebice su važne respiratorne
alergijske reakcije kod djece koje sve češće prerastaju u kronične oblike ili
prelaze u astmu te alergijske reakcije koje se javljaju kao profesionalna
oboljenja. Jedan od glavnih ciljeva u znanstvenim istraživanjima alergija je
iznalaženje novih dijagnostičkih alata alergijskih bolesti kao i novih mogućnosti
za specifičnu imunoterapiju.
U okviru ovog diplomskog rada pripravljene su i karakterizirane nanolipidne
formulacije proteina izoliranih iz peluda biljke Ambrosia elatior. Formulacije su
temeljene na liposomima s ugrađenim proteinima, koji su inkorporirani u gelsku
mrežu peptidnog hidrogelatora Ac-L-Phe-L-Phe-L-Ala-NH2. Metodom hidratacije
tankog lipidnog filma pripremljeni su multilamelarni liposomi. Efikasnost
ugrađivanja proteina iz peluda A. elatior u liposome određena je
spektrofotometrijski i iznosila je ~13,01%. Priređene liposomske formulacije
alergena su karakterizirane metodom dinamičkog raspršenja svjetlosti i
pokazano je kako ugrađivanje proteina A. elatior dovodi do povećanja liposoma i
povećanja negativnog površinskog naboja. Ispitana su i gelirajuća svojstva
hidrogelatora Ac-L-Phe-L-Phe-L-Ala-NH2 uz dodatak liposoma. Pokazano je da
liposomi ne narušavaju gelsku mrežu hidrogelatora pri koncentraciji lipida manjoj
od koncentracije gelatora. Ugrađivanje proteina Ambrosia elatior, te modelnog
proteina albumina (BSA) / FITC-BSA rezultiralo je sporijim nastajanjem gela ili
nemogućnošću prijelaza u gel.
Sustavi liposomskih formulacija modelnog proteina BSA i FITC-BSA
inkorporiranih u hidrogel su karakterizirani elektronskom (TEM) i konfokalnom
mikroskopijom. Transmisijskom elektronskom mikroskopijom potvrđeno je
ugrađivanje liposoma u hidrogel čija je morfologija ostala ista. U uzorcima gela s
ugrađenim BSA, gelska mreža ostala je očuvanog integriteta, no uočeno je kako
su gelske niti bile tanje i gušće. Konfokalna mikroskopija potvrdila je ugrađivanje
FITC-BSA u liposomima, u hidrogel.Allergic reactions (hypersensitivity reactions) represent a significant medical
problem of modern society and lifestyle. Of particular importance are respiratory
allergic reactions in children, which are increasingly becoming chronic or
progressing into asthma, and allergic reactions occuring as occupational
diseases. One of the main goals in scientific research of allergies is finding new
diagnostic tools for allergic diseases as well as new possibilities for specific
immunotherapy.
For this graduate thesis nanolipid formulations of proteins isolated from the
Ambrosia elatior pollen were prepared and characterized. The formulations are
based on liposomes with embedded proteins that are incorporated into the gel
network of the peptide hydrogelator Ac-L-Phe-L-Phe-L-Ala-NH2. Multilamellar
liposomes were prepared using the method of thin lipid film hydration. Efficacy of
incorporating A. elatior proteins into liposomes was spectrophotometrically
determined to be approx. 13.01%. Prepared liposomal formulations of the
allergens were characterized by dynamic light scattering method. It was
demonstrated that incorporation of A. elatior proteins leads to an increase in
liposomes and an increase in negative surface charge. The gelling properties of
the hydrogelator Ac-L-Phe-L-Phe-L-Ala-NH2 with addition of liposomes were also
studied. It was shown that liposomes do not impair the gel network of
hydrogelator at lipid concentrations lower than the gelator concentration. The
incorporation of Ambrosia elatior proteins and model protein albumin (BSA) /
FITC-BSA resulted in slower gel formation or inability to transition into gel.
BSA and FITC-BSA liposome formulations incorporated in the hydrogel were
characterized by electron (TEM) and confocal microscopy. Transmission electron
microscopy confirmed the incorporation of liposomes into the hydrogel. In gel
samples with built-in BSA, the gel network preserved integrity, but it was noticed
that the gels fiber were thinner and thicker. Confocal microscopy proved the
incorporation of FITC-BSA into liposomes in the hydrogel
Azithromycin and chlorine e6 conjugate synthesis study
No full textU ovom radu opisan je razvoj sinteze novoga konjugata azitromicina i klorina e6. Pripravljeni derivat azitromicina s aktiviranom 4''-O-acilimidazolilnom funkcionalnom skupinom i 13¹-etilendiaminski derivat klorina e6 su reakcijom amidnog vezanja u završnom stupnju paralelne sinteze povezani u željeni konjugat. Svim izoliranim spojevima strukture su potvrđene i okarakterizirane 1H i 13C NMR, UV/Vis i IR spektrima a završnom spoju 21 dodatno i 2D spektroskopijom COSY, NOESY i TOCSY. Svrha je sintetiziranih spojeva ispitivanje mogućnosti njihova korištenja u antimikrobnoj fotodinamičkoj terapiji.This work describes the synthesis of a new conjugate of azithromycin and chlorine e6. The prepared azithromycin derivative with the activated 4"-O-acylimidazolyl functional group and the 13-ethylenediamine derivative of chlorine e6 are reacted with the amide bond in the final step of the parallel synthesis linked to the desired conjugate. All isolated structural compounds were confirmed and characterized by 1H and 13C NMR, UV/Vis and IR spectra and final compound 21 in addition to 2D COSY, NOESY and TOCSY spectroscopy. The purpose of the synthesized compounds is to investigate the possibility of their use in antimicrobial photodynamic therapy
Utjecaj iona željeza na preživljavanje i razmnožavanje F.novicida i F.tularensis subsp. Holartica sojLVS u vodi
No full textFrancisella tularensis je fakultativni, aerobni, gram-negativni unutarstanični patogen i uzročnik je bolesti tularemije, u ljudi i životinja. Pripada razredu Gammaproteobacteria, obitelji Francisellaceae te rodu Francisella. Vrsti tularensis pripada podvrsta tularensis ili Tip A, podvrsta holarctica ili Tip B te podvrsta mediasiatica dok novicida čini zasebni rod. Cilj rada bio je odrediti utjecaj različitih koncentracija iona željeza (0.1 μM; 0.8 μM; 0.1 mM; 0.5 mM i 1.0 mM) na preživljavanje i razmnožavanje F. novicida i F. tularensis subsp. holarctica, soj LVS, u redestiliranoj vodi. Broj bakterija (CFU/mL) određen je nakapavanjem serijskih razrijeđenja na hranjive podloge svakih 24 sata tijekom 5 dana. Rezultati su pokazali da prisustvo željeza u najvećim koncentracijama (0.1 mM; 0.5 mM i 1.0 mM) u redestiliranoj vodi bitno smanjuje razmnožavanje bakterija. Koncentracija od 0.8 μM iona željeza pokazala se optimalnom za preživljavanje bakterija u redestiliranoj vodi. Također se dokazalo iskorištenje željeza od strane F. novicida i F. tularensis subsp. holarctica, soj LVS, analizom otopina željezovih iona prije (0. dan) i 5. dan nakon inokulacije bakterija pomoću atomskog spektrometra