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Zašto srce gotovo nikad ne obolijeva od tumora?
No full textCardiac tumors, both primary tumors and metastasis are extremely rare.
If this is intuitive for cardiomyocytes, which early after birth exit the cell
cycle and terminally stop dividing, the heart contains many other cell
types which have the potential to proliferate. Angiogenesis is a key
causative factor in the pathogenesis of any tumor, as in the absence of
vascular support tumors become necrotic and apoptotic.
The main goal of this project was to compare tumor growth and
angiogenesis in the myocardium and in the right tibialis anterior muscle.
Mices were subjected to intracardiac and intramuscular injection of
different quantities of Lewis lung carinoma cells. Tumor masses were
harvested and used for immunohistochemistry, immunofluorescence and
dextran assays. Results show higher tumor cell growth in the muscular
tissue compared to the cardiac tissue, higher percentage of positive
αSMA/lectin tumor cells, as well as higher percentage of Edu positive cells
in muscular tumor tissues compared to the untreated tissue. Results from
dextran assays show higher vascular permeability and perfusion of the
cardiac tumor compared to the muscle.
This project provide evidence that experimental tumors in the heart are
less invasive then tumor in the muscle, although the main reason why
cardiac tumor almost never occur still remain unexplored.Srčani tumori, kako primarni tako i metastatski, su iznimno rijetki. Iako se
kao glavni razlog toga smatraju kardiomiociti koji rano nakon rođenja
izlaze iz staničnog ciklusa i trajno se prestaju dijeliti, postoje i druge
srčane stanice koje imaju potencijal proliferacije. Angiogeneza je ključni
faktor u patogenezi bilo kojeg tumora, s obzirom da u odsutsvu
vaskularne potpore tumor postaje nekrotički i apoptotski.
Glavni cilj ovog projekta bio je usporediti rast tumora i angiogenezu u
miokardu i prednjem potkoljeničnom mišiću desne noge.
U miševe su intrakardijalno i intramuskularno injektirane različite
koncentracije Lewis lung tumorskih stanica. Nakon određenog perioda
tumori su izvađeni i dalje korišteni za imunohistokemijsku,
imunofluorescenciju i analizu pomoću dekstrana.
Dobiveni rezultati su pokazali veći broj tumorskih stanica u mišićnom tkivu
u odnosu na srčano tkivo, veći postotak pozitivnih αSMA / lektin tumorskih
stanica, kao i veći postotak Edu pozitivnih stanica u mišićnim tumorskim
tkivima u usporedbi s netretiranim tkivom. Rezultati dekstranskih testova
pokazuju veću permeabilnost i perfuziju srčanog tumora u odnosu na
mišićni.
Eksperimentalni podatci ovog projekta dokazuju da je srčani tumor manje
invazivan od mišićnog, ali glavni razlog zašto srce gotovo nikad ne
obolijeva od tumora ostaje nepoznat
Influence of antibiotic combinations on Escherichia coli in Luria-Bertani and minimal M9 media
No full textInfekcije urinarnog trakta (URI) koje uzrokuju Escherichia coli (E. coli) postaju sve veći problem diljem svijeta. Cilj rada je istražiti da li postoji učinkovit način da se infekcije brže izliječe sa terapijom kombinacije antibiotika. Da bi mogli napraviti istraživanje učinka kombinacije antibiotika bilo je potrebno odrediti minimalnu inhibicijsku koncentraciju (MIK) za antibiotike ciprofloksacin, rifampicin i streptomicin. Svaki od njih ima različit mehanizam djelovanja. MIK za ciprofloksacin iznosi 12,5
μg/ml, rifampicin 100 μg/ml i streptomicin 25 μg/ml. MIK vrijednosti antibiotika koje smo odredili eksperimentalno slažu se sa literaturnim vrijednostima. Kombinacije antibiotika koje smo koristili u ovom radu sastavljene su od koncentracija MIK pojedinačnih antibiotika. Istražili smo djelovanje još jednog važnog čimbenika, hranjivog medija u kojem se nalaze. Koristila su se dva medija, Luria – Bertani koji je bogat hranjivim tvarima i minimalni M9 medij koji je siromašan hranjivim tvarima. Usporedbom krivulja rasta u prisutnosti kombinacije antibiotika uočili smo da bakterije prestanu rasti nakon 1 h inkubacije sa svim testiranim kombinacijama te vidimo sličan broj bakterija tijekom 24 h uzorkovanja u LB mediju dok u M9 mediju primjećujemo blagi rast stanica tijekom inkubacije. Usporedba krivulja smrtnosti pojedinačnih antibiotika i njihovih kombinacija u LB mediju pokazala je brži i značajno veći pad broja bakterijskih stanica kod kombinacije antibiotika unutar 24 h. Slično djelovanje u oba medija ima kombinacija rifampicin – streptomicin, dok ostale kombinacije pokazuju sporiji baktericidni učinak. Usporedbom svih MIK koncentracija antibiotika u LB mediju, vidimo da imaju slično djelovanje no za sve testirane koncentracije primjećujemo da rifampicin ima najveću učinkovitost. Metodom antibiograma se pokazalo da kombinacija ciprofloksacin - streptomicin ima najučinkovitije djelovanje u LB i M9 mediju te je pokazala najveći porast učinka u usporedbi s pojedinačnim antibioticima od čak 20 puta. Usporedba sa antibiogramom je pokazala da većina koncentracija pojedinačnih antibiotika ciprofloksacina i streptomicina su pokazale jače djelovanje u LB mediju dok je kombinacija ciprofloksacin – streptomicin pokazala puno bolju učinkovitost u M9 mediju. Rezultate koje smo dobili eksperimentalnim radom bi se mogli iskoristiti kao temelj za buduća istraživanja rezistencije.Escherichia coli (E. coli) urinary tract infections (UTIs) are becoming an increasing problem all over the world. The aim of this thesis was to find out whether there is an effective way to cure these infections faster with therapy combining different antibiotics. To be able to make a research about effects of antibiotic combinations, it was necessary to determine the minimum inhibitory concentration (MIC) for antibiotics: ciprofloxacin, rifampicin and streptomycin. Each of them has a different mechanism of action. Ciprofloxacin MIC is 12.5 μg/ml, rifampicin 100 μg/ml and streptomycin 25 μg/ml. The antibiotic MIC values that have been determined experimentally agree with the literary values. The antibiotic combinations used in this thesis were composed of the MIC concentrations of each individual antibiotic. Besides the research of the effect of antibiotics combination, the nutrient media they are part of has also been researched. Two different media were used: Luria - Bertani, media rich in nutrients, and a minimal M9 medium that is poor in nutrients. By comparing growth curves in the presence of antibiotic combination, it was observed that bacteria ceased to grow after 1 h incubation with all tested combinations. Also, a similar number of bacteria for 24 h of LB media sampling was noticed, while in M9 media slight growth of cells during incubation was observed. Furthermore, by comparison of kill curves of individual antibiotics and their combinations in LB medium, a faster and significantly higher bacterial cell count decrease in antibiotic combination within 24 h was observed. Similar action in both media was detected with combination rifampicin - streptomycin, while the other combinations showed a slower bactericidal effect. By comparison of all MIC antibiotic concentrations in LB media, it was seen that they have similar activity, but for all tested concentrations rifampicin demonstrated the highest efficacy. The antibiogram method showed that the ciprofloxacin - streptomycin combination had the major effect on LB and M9 media, and showed the highest effect compared to single antibiotics; up to 20 times. Comparison of antibiograms revealed that individual antibiotics ciprofloxacin and streptomycin, regardless their concentrations, had higher activity in the LB medium, while their combination showed better efficacy in the M9 medium. These results obtained by experimental work demonstrated efficacy of combined antibiotics therapy and therefore, set a basis for future research of bacterial resistance
Disrupted in Schizophrenia 1 regulates the processing of reelin in the perinatal cortex
Get PDFDisrupted in Schizophrenia 1 (DISC1) is a prominent gene in mental illness research, encoding a scaffold protein known to be of importance in the developing cerebral cortex. Reelin is a critical extracellular protein for development and lamination of the prenatal cortex and which has also been independently implicated in mental illness. Regulation of reelin activity occurs through processing by the metalloproteinases ADAMTS-4 and ADAMTS-5. Through cross-breeding of heterozygous transgenic DISC1 mice with heterozygous reeler mice, which have reduced reelin, pups heterozygous for both phenotypeswere generated. Fromthese,we determine that transgenic DISC1 leads to a reduction in the processing of reelin, with implications for its downstream signalling element Dab1. An effect of DISC1 on reelin processing was confirmed in vitro, and revealed that intracellular DISC1 affects ADAMTS-4 protein, which in turn is exported and affects processing of extracellular reelin. In transgenic rat cortical cultures, an effect of DISC1 on reelin processing could also be seen specifically in early, immature neurons, but was lost in calretinin and reelin-positive mature neurons, suggesting cell-type specificity. DISC1 therefore acts upstream of reelin in the perinatal cerebral cortex in a cell type/time specific manner, leading to regulation of its activity through altered proteolytic cleavage. Thus a functional link is demonstrated between two proteins, each of independent importance for both cortical development and associated cognitive functions leading to behavioural maladaptation and mental illness
Synthesis and properties of novel (oxido)pyridyl porphyrins for use in photodynamic therapy
No full textFotodinamička terapija (PDT) predstavlja jedan od suvremenijih pristupa liječenju tumora. Komponente fotodinamičke terapije su fotosenzibilizator (PS), kisik i svjetlo, tri zasebno netoksične kompone, koje zajedno stvaraju toksičan učinak na stanice. Za uspješnu fotodinamičku terapiju bitan je odabir fotosenzibilizatora kao i njegova lokalizacija u stanicama.
Porfirini, tetrapirolni makrociklički spojevi, zbog svojeg karakterističnog apsorpcijskog i emisijskog spektra, kao i visokog prinosa singlet kisika, predstavljaju vrlo učinkovite fotosenzibilizatore za PDT. Amfifilni porfirini su vrsta porfirina koji se sastoje od hidrofilnog dijela koji omogućuje bolju topljivost u vodi, te hidrofobnog dijela koji služi lakšem prolasku kroz staničnu membranu. Sinteze amfifilnih porfirina su relativno jednostavne, no problem predstavlja pročišćavanje dobivenih spojeva. U sintezi (oksido)piridilporfirina konjugiranih lancima masnih kiselina, kao glavni problem se pokazala N-oksidacija piridilnih dušikovih atoma, odnosno pročišćavanje produkata.
U ovom radu optimizirane su reakcije N-oksidacije različitih 3-piridilporfirina te ispitani alternativni putevi sinteze novih (oksido)piridilporfirina. Sintetiziranim porfirinima su ispitana fotofizikalna svojstva, te je utvrđeno da svi sintetizirani piridilporfirini imaju visok prinos singletnog kisika.
Testom internalizacije (oksido)piridilporfirina primijećen je problem netopljivosti dobivenih spojeva u vodi i puferima, te je došlo do agregacije spoja na staničnoj membrani i spoj nije internalizirao u stanicu. Također, pokazalo se da je spoj vrlo toksičan i pri malom intezitetu svjetla zbog nevijabilnog izgleda stanica tijekom testa.Photodynamic Therapy (PDT) is one of the most recent approaches to treating tumors. Components of the PDT are photosensitizer (PS), oxygen and light, three non-toxic components that together can produce a toxic effect on the cells. For successful photodynamic therapy, it is essential to select a photosensitizer with good localization in cells.
Porphyrins, tetrapyrrole macrocyclic compounds, due to their specific absorption and emission spectra, as well as the high yield of singlet oxygen, represent a highly efficient photosensitizers for PDT. Amphiphilic porphyrins are type of porphyrins that consist of hydrophilic part that allows better water solubility and hydrophobic part of the molecule that facilitates passing through the cell membrane. The synthesis of amphiphilic porphyrins is relatively simple, however purification of the compounds is often very difficult. In the synthesis of (oxidopyridyl)porphyrins conjugated with fatty acid chains, the main problem that occured was N-oxidation of the pyridyl nitrogens, and purification of the products.
In this work, attempts for optimization of N-oxidation reactions of various 3-pyridylporphyrins and alternative pathways of synthesis of new (oxidopyridyl)porphyrin are described. Photophysical properties of the synthesized porphyrins were tested, and it was found that all synthesized pyridyporphyrins produce singlet oxygen in high yields.
In the internalization test it was found that synthesized (oxido)pyridylporphyrin was not sufficiently soluble in water and buffers, and did not internalize into the cell probably due to the aggregation of the compound on the cell membrane. Also, it was shown that the compound is very toxic even with low intensity of light used for photoactivation, as shown by appearance of non-viable cells during the test
Neurobiologija ljubavi
No full textNeurobiologija ljubavi je područje neuroznanosti koje istraţuje molekularnu i staničnu
osnovu ljubavi, odnosno ulogu hormona, neurotransmitera i gena u njenom nastajanju i
modulaciji. Ovo je novo područje neuroznanosti koje se razvilo u nekoliko prošlih desetljeća,
u kojima su znanstvenici intenzivno istraţivali promjene u mozgu vezane uz osjećaj ljubavi,
koristeći najsuvremenije metode istraţivanja kao PET, fMRI, te genetske, biokemijske i
psihološke tehnike istraţivanja. U ovom radu detaljno su opisani modeli, rezultati i
metodologija ovih istraţivanja, te molekule, geni i receptori za koje se smatra da su ključni u
stvaranju osjećaja ljubavi. Nadalje, prikazana su područja mozga koja su mjesta aktivacije i
deaktivacije hormona, gena i ostalih molekula koje se mijenjaju u različitim fazama ljubavi.
Ovo su prve spoznaje o molekularnim i staničnim mehanizmima koji su osnova ljubavi, a
daljnja istraţivanja u ovom području pojasnit će nam jedan od temeljnih osjećaja na kojem se
zasniva opstanak ţivotinjskih vrsta i čovjeka
Allysiy of prp expression in Mycobacterium smegmatis in presence of propionate by using fluorescent time-lapse microscopy
No full textFenotipska heterogenost može imati važne funkcionalne posljedice, može
pružiti pojedincima i skupinama novu funkcionalnost, te omogućiti
preživljavanje u stresnim uvjetima poput nedostatka kisika i hranjivih
tvari ili utjecaja antibiotika. Pojava rezistentnosti u postojećoj
antituberkuloznoj terapiji, potaklo je da poznavanje fenotipske
heterogenosti postane važni istraživački fokus. Mycobacterium smegmatis
je brzorastuća nepatogena bakterija koja služi kao modelni organizam za
patogen Mycobacterium tuberculosis koji je glavni uzročnik tuberkuloze.
Za vrijeme infekcije posebno je važan metabolizam masnih kiselina u
mikobakterijama. Potreba za razumijevanjem metabolizma masnih
kiselina s neparnim brojem C atoma, metilcitratnom ciklusu i važnosti prp
gena potaklo je istraživanja na mediju koji sadrži propionat. Korištenjem
time-lapse fluorescentne mikroskopije i obrade filmova u BactImAS
platformi omogućeno je proučavanje ekspresije prp gena i drugih
parametara tijekom vremena, te uočavanje fenotipske heterogenosti u
našim eksperimentima. Rezultati su analizirani po principu stanica po
stanica (eng. cell-to-cell analysis) i kolektivnom (eng. bulk) analizom. U
ovom radu, u pet eksperimenta s istim uvjetima, zapažena je
heterogenost M. smegmatis, odnosno pojedinačne se stanice međusobno
razlikuju s obzirom na ekspresiju gena i drugih fenotipskih osobina.
Uočena je razlika u brzini prilagodbe i jačini zelene fluorescencije koja
odgovara ekspresiji prp gena u pojedinačnim stanicama unutar istog
eksperimenta. Sestrinske stanice pokazuju razliku i u brzini rasta i diobe,
veličini površine stanice i broju potomaka. U većini slučaja pokazalo se da
jedna sestrinska stanica brže raste, prije se podijeli i ima jaču ekspresiju
gena. S obzirom na dokazanu heterogenost nije moguće postaviti točne
obrasce i pravila koja vrijede za ekspresiju prp gena u prisutnosti
propionata. Nije dokazana povezanost ekspresije gena s veličinom stanica,
brzinom rasta, brojem potomaka i ostalih ispitivanih parametara.
BactImAS softver je omogućio veliki napredak u analizi mikobakterija.
Aktivnim korištenjem program je unaprijeđen i ispravljeni su nedostaci.
Ovim radom otvaraju se mnogobrojna pitanja, te potreba za novim
analizama i iskorištavanjem BactImAS-a u proučavanju mikobakterija i
heterogenosti.Phenotypic heterogeneity can have important functional consequences, it
can provide individuals and groups with new functionality, and it can also
enable survival in stressful conditions such as lack of oxygen and nutrients
or antibiotic influence. The emergence of resistance to existing
antituberculous therapy has led to the recognition of phenotypic
heterogeneity as an important research focus. Mycobacterium smegmatis
is a rapidly growing non-pathogenic bacterium that serves as a model for
the pathogen Mycobacterium tuberculosis, which is the major cause of
tuberculosis. During the infection, fatty acid metabolism in mycobacteria is
particularly important. Understanding fatty acid metabolism with odd
number of C atoms, methylcitrate cycle and the importance of the prp
gene stimulated studies on a propionate-containing media. Using timelapse
fluorescent microscopy and movie processing on BactImAS platform,
it was possible to study the expression of prp genes and other parameters
in time and to observe phenotypic heterogeneity in our experiments. The
results were analyzed by cell-to-cell analysis and bulk analysis. In this
thesis, in five experiments with the same conditions, the heterogeneity of
M. smegmatis was noticed, meaning that the individual cells differed with
respect to the expression of genes and other phenotypic traits. There was
a difference in the rate of adjustment and the strength of green
fluorescence, that corresponds to prp expression level, in individual cells
within the same experiment. Sister cells show the difference in the rate of
growth and division, cell surface size and number of offspring. In most
cases it has been shown that one sister cell inherits faster growth, shorter
division time and it has a stronger gene expression. Given the proven
heterogeneity, it is not possible to set the exact patterns and rules that
apply to the expression of the prp gene in the presence of propionate. No
correlation of genetic expression with cell size, growth rate, number of
offspring and other investigated parameters has been demonstrated.
BactImAS software is a great discovery for mycobacterial analysis. Active
usage of the program has been improved and shortcomings are corrected.
This work opens up many questions, and the need for new analyses and
the exploitation of BactImAS in the study of mycobacteria and
heterogeneity
Razumijevanje slabe sposobnosti angiogeneze odraslog srca
No full textAngiogenesis is the process of new blood vessel formation, crucial for the development of an embryo as well as for organ growth and wound healing later in the adulthood. A key factor responsible for the stimulation of angiogenesis is VEGF165, the most potent splicing isoform of VEGF-A. Based on recent results obtained in the host laboratory, the driving hypothesis standing beyond this work was to investigate why endothelial cells in the skeletal muscle and in the neonatal heart respond to AAV-VEGF165 inducing angiogenesis, whereas in the adult heart they do not. For this purpose, we investigated the responsiveness of adult cardiac and neonatal endothelial cells (ECs), as well as of muscle ECs to the proangiogenic factor VEGF in vitro. Based on the observation that cardiac ECs lose their proliferative capacity at day 7 after birth, we performed a transcriptome analysis of the three EC populations and identified a few differentially expressed genes (DEGs). Particularly, we focused on Dhcr24, the most interesting gene among DEG between pre-natal and post-natal ECs. We developed a genome editing strategy to investigate its functional role in angiogenesis. In the first set of experiments, the proliferation of adult and neonatal cardiac ECs and muscle ECs was assessed after VEGF administration at different time points, followed by immunofluorescence staining and image acquisition. Interestingly and different from their in vivo behaviour, adult cardiac ECs proliferated in response to VEGF at both time points, suggesting that some inhibitory factor might block their response in the adult heart in vivo. In the second set of experiments, the CRISPR/Cas9 technology was used to silence Dhcr24 in SVEC and LG cell lines. Successful knockout was confirmed by the downregulation of Dhcr24 protein expression in Western blot. Development of an efficient genome editing strategy could offer an opportunity to genetically modify Dhcr24 in quiescent ECs and test its ability to restore the pro-angiogenic phenotype.Angiogeneza je proces nastajanja novih krvnih žila, ključan tijekom razvoja embrija, te rasta organa i zacjeljivanja rana u odrasloj dobi. Ključni čimbenik koji je odgovoran za stimulaciju angiogeneze je VEGF165, najpotentnija izoforma alternativnog prekrajanja VEGF-A. Na temelju nedavno dobivenih rezultata u našem laboratoriju, glavna hipoteza ovog rada bila je istražiti zašto endotelne stanice (ES) u skeletnim mišićima i u neonatalnom srcu reagiraju na AAV-VEGF165 induciranu angiogenezu, dok one u odraslom srcu ne. U tu smo svrhu istražili odgovor odraslih i neonatalnih srčanih ES, kao i mišićnih ES mišića na proangiogeni čimbenik VEGF in vitro. Na temelju opažanja da srčane ES gube sposobnost proliferacije 7. dan nakon rođenja, izvršili smo transkripcijsku analizu tri populacije ES i identificirali nekoliko diferencijalno eksprimiranih gena (DEG). Posebno smo se usredotočili na Dhcr24, najzanimljiviji gen između DEG između prenatalnih i postnatalnih ES. Razvili smo strategiju uređivanja genoma kako bismo istražili njegovu funkcionalnu ulogu u angiogenezi. U prvoj skupini eksperimenata, proliferacija odraslih i neonatalnih srčanih ES i mišićnih ES određena je nakon administracije VEGF-a u različitim vremenskim točkama, nakon čega je slijedila imunofluorescencija i mikroskopija. Zanimljivo, za razliku od ponašanja in vivo, odrasle srčane ES su proliferirale kao odgovor na VEGF u obje vremenske točke, sugerirajući prisustvo nekog inhibitornog čimbenika koji može blokirati odgovor na VEGF u odraslom srcu in vivo. U drugoj skupini eksperimenata, CRISPR/Cas9 tehnologija korištena je za utišavanje Dhcr24 u SVEC i LG staničnim linijama. Uspješan knockout potvrđen je smanjenjem ekspresije Dhcr24 proteina u Western blotu. Razvijanje učinkovite strategije za uređivanje genoma moglo bi pružiti priliku za genetsko modificiranje Dhcr24 u ES u stanju mirovanja te ispitati ima li sposobnost vraćanja proangiogenog fenotipa
Analysis of potential drugs for Alzheimer's disease on human neuroblastoma cells
No full textZnačaj: Alzheimerova bolest je najčešći oblik demencije, koja trenutno
predstavlja najskuplju stavku u sustavu zdravstvene zaštite u razvijenim
zemljama. Alzheimerova bolest je ireverzibilni, progresivni
neurodegenerativni poremećaj karakteriziran postepenim gubitkom
pamćenja i kognitivnih vještina. Poremećaj u metabolizmu Aβ peptida
smatra se centralnim mehanizmom nastanka Alzheimerove bolesti. Za sada
postoji samo neučinkovito simptomatsko liječenje te stoga farmaceutska
industrija ulaže velike napore kako bi se pronašao lijek koji bi usporio
napredak ili spriječio nastanak Alzheimerove bolesti. Zbog toga je jedna od
glavnih meta u istraživanju lijekova za Alzheimerovu bolest
intramembranska proteaza γ-sekretaza koja stvara toksične Aβ peptide.
Rezultati: Prvo smo optimizirali sandwich ELISA metodu za kvantifikaciju
unutarstaničnog Aβ 1-40 peptida pri niskim fiziološkim koncentracijama
kako ne bi morali koristiti genetski modificirane SH-SY5Y stanice.
Optimizirani visoko osjetljivi ELISA testovi korišteni su za ispitivanje
potencijalnih lijekova tvrtke JIVA Pharma. Svi ispitani JIVA spojevi pokazali
su inhibiciju aktivnosti γ-sekretaze u koncentracijama od 10 μM i 100μM te
ni jedan spoj nije promijenio morfologiju stanica ili uzrokovao njihovu smrt.
U koncentraciji od 10 μM, najveću inhibiciju pokazao je spoj JIVA 025, dok
su u koncentraciji od 100 μM, najveću inhibiciju pokazali JIVA 024 i JIVA
038. Iako su spojevi JIVA 025, JIVA 027 i JIVA 042 pokazali inhibiciju
aktivnosti γ-sekretaze veću od 50% u obje ispitivane koncentracije, razlika
u jačini inhibicije nije bila značajna.
Zaključak: Spojevi JIVA 024 i JIVA 038 pokazali su značajnu razliku u
inhibiciji aktivnosti γ-sekretaze kada su korišteni u različitim
koncentracijama te imaju dobar potencijal za daljnju optimizaciju strukture.Significance: Alzheimer's disease is the most common form of dementia,
currently the most expensive element in the healthcare system in developed
countries. Alzheimer's disease is an irreversible, progressive
neurodegenerative disorder characterized by gradual loss of memory and
cognitive skills. The disorder in the metabolism of Aβ peptide is considered
to be the central mechanism of Alzheimer's disease. For now, only
ineffective symptomatic treatment is available and therefore the
pharmaceutical industry is making great efforts to find a drug that would
slow down progress or prevent the onset of Alzheimer's disease. Therefore,
one of the main targets in drug research for Alzheimer's disease is the
intramembrane protease γ-secretase which produces toxic Aβ peptides.
Results: We first optimized the sandwich ELISA method for quantification
of the intracellular Aβ 1-40 peptide in low physiological concentrations, to
avoid use of genetically modified SH-SY5Y cells. Optimized high-sensitivity
ELISA tests were used to investigate potential drugs from JIVA Pharma
company. All of the tested JIVA compounds showed inhibition of γ-secretase
activity at concentrations of 10 μM and 100 μM, and none of the compounds
changed the cell morphology or caused their death. At a concentration of
10 μM, the highest inhibition was shown by the compound JIVA 025, while
at a concentration of 100 μM, the highest inhibition was shown by JIVA 024
and JIVA 038. Although compounds JIVA 025, JIVA 027 and JIVA 042
showed inhibition of γ-secretase activity greater than 50% in both tested
concentrations, differences in inhibition strength were not significant.
Conclusion: Compounds JIVA 024 and JIVA 038 showed a significant
difference in inhibition of γ-secretase activity when used at different
concentrations and showed potential for further structure optimization
Sinteza borovog kompleksa dipirometena za konjugaciju s proteinima
No full textBorov kompleks dipirometena (BODIPY®) predstavlja razred fluorescentnih boja koje se mogu koristiti u medicinske i biološke svrhe. Fluorescentno obilježavanje molekula nastaje konjugacijom, odnosno vezanjem fluorescentnih boja za određene proteine i peptide, s ciljem praćenja njihovih interakcija, konformacijskih promjena i lokalizacije pojedinih peptida i dijelova proteina. BODIPY molekule su prilično neosjetljive na polarnost i pH okoliša te vrlo stabilne u fiziološkim uvjetima. Jezgra BODIPY fluorescentnih boja je dovoljno snažna da izdrži niz kemijskih promjena, a male promjene u strukturi omogućuju aktivaciju fluorescentnih svojstava. U ovom radu uspješno je sintetiziran N,N'-difluoroboril-2,8-dietil-1,3,7,9-tetrametil- 5-(4-izotiocijanatofenil)-dipirometen, BODIPY derivat koji koristi za konjugaciju s proteinima. Opisan je alternativan put sinteze u odnosu na literaturni. Iako je prilikom sinteze došlo do gubitka fluorescentne skupine, na kraju se uspio dobiti željeni BODIPY te su raspravljeni mogući putevi sinteze u kojima bi fluorescentna skupina ostala očuvana.The boron complexes of dipyrromethene (BODIPY®) comprise a class of fluorescent dyes that can be used in medical and biological studies. Fluorescent molecular labeling can be obtained by conjugation, or linking fluorescent dyes to certain proteins and peptides, in order to detect interactions, conformational changes and localization of specific peptides and parts of a protein. BODIPY molecules are relatively insensitive to the polarity and pH of their environment and are very stable to physiological conditions. The BODIPY core is strong enough to withstand a series of chemical reactions, and even the smallest changes to their structures enable the activation of their fluorescent properties. Herein, we have successfully synthesized N, N'-difluoroboryl-2,8-diethyl-1,3,7,9-tetramethyl-5-(4-isothiocyanatophenyl)-dipyrromethene, BODIPY derivative that is used for conjugation to proteins. Alternative pathway of synthesis is described with regard to literature. During the synthesis, a fluorescent group was lost in one step, however, desired BODIPY has been synthesized, thus possible pathways of synthesis in which fluorescent group can be preserved are discussed
Defining the preferential consumption of cocaine and methamphetamine in Drosophila melanogaster
No full textOvisnost je stanje u kojem organizam osjeća jaku psihičku ili fizičku
potrebu za opojnom supstancom. Psihostimulativne droge, poput kokaina i
metamfetamina, djeluju na centre u mozgu koji reguliraju procesuiranje
nagrade. Nagrađujući utjecaj droga kao posljedicu ima povećanu privlačnost
droge koja dovodi do ovisničkog ponašanja, a može se ispitivati na
laboratorijskim životinjama kao preferencijalna konzumacija. Iako postoje mnogi
eseji za mjerenje lokomotornih efekta kokaina i metamfetamina, platforma
kojom bi se promatrao nagrađujući utjecaj psihostimuansa kod Drosophile
melanogaster dosada nije bila razvijena.
Cilj ovog diplomskog rada bio je modificirati Capillary Feeding (CAFE) esej
prvotno osmišljen za ispitivanje ovisnosti o alkoholu i definirati karakteristike
preferencijalne konzumacije kokaina i metamfetamina kod vinske mušice kao
modelnog organizma za izučavanje ovisničkih ponašanja. Utvrdili smo kako
mušice prilikom samoadministracije kokaina i metamfetamina razvijaju obrasce
ovisničkog ponašanja primijećene u ljudi i modelima glodavaca. One aktivno
biraju i preferiraju hranu s kokainom ili metamfetaminom unatoč gorkom okusu
ovih psihostimulansa, te nastavljaju konzumaciju usprkos negativnim
posljedicama i nakon deprivacije razvijaju ponašanja slična recidivu. Naši
rezultati sugeriraju kako su u razvoju preferencijalne konzumacije na kokain
uključeni produkti cycle gena, u preferenciji metamfetamina produkti period,
Clock i cycle gena, te kako dopaminski transporter i receptor tip 1 nisu uključeni
u razvoj preferencijalne konzumacije.
Korištenje Drosophile u ispitivanjima samoadministracije omogućiti će
daljnja istraživanja neuroloških mehanizama koji su u podlozi nagrađujućeg
djelovanja psihostimulansa kako bi se u budućnosti dizajnirali farmakološki
tretmani za spriječavanje ili liječenje ovisnosti.Addiction is a condition where organism feels a strong psychological or physical
need for narcotic substance. Psychostimulative drugs, like cocaine and
methamphetamine, affect neural mechanisms involved in the processing of
rewards. Because of the rewarding effect of a drug, attraction for a drug leads to
addictive behavior. This behavior can be investigated in laboratory animals as
preferential consumption. Although there are many assays to measure locomotor
effects of cocaine and methamphetamine, a way to measure rewarding influence
of psychostimulants in Drosophila melanogaster has not yet been developed.
The aim of this thesis was to define the characteristics of preferential
consumption for cocaine and methamphetamine in fruit flies as a model organism
to study addictive behaviors. In order to do that, we modified the Capillary
Feeding (CAFE) essay initially designed to test alcohol dependence. We find that
fruit flies willingly self-administrate cocaine and methamphetamine and develop
patterns of addictive behavior similar to ones observed in humans and rodent
models. Fruit flies actively choose and prefer food with cocaine or
methamphetamine despite of bitter taste of these psychostimulants, they
continue to consume drugs despite negative consequences and after deprivation
develop behaviors similar to relapse. Our results suggest that the development of
preferential consumption for cocaine depends on cycle gene products, that
preference for methamphetamine involves period, Clock and cycle genes, and
that dopamine transporter and dopamine receptor type 1 are not involved in the
development of preferential consumption.
The use of Drosophila in self-administration experiments will be useful in further
research on neurological mechanisms of rewarding action of psychostimulants,
with final goal of designing pharmacological treatments for prevention and
treatment of addiction