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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Razvoj bioloških testova za identifikaciju liganada steroidnih receptora i ispitivanje aktivnosti steroidogenog enzima aromataze / Development of biological assays for identification of steroid receptor ligands and determination of activity of steroidogenic enyzme aromatase

Bekić Sofija 07 August 2020 (has links)
<p>U ovoj doktorskoj disertaciji&nbsp; razvijen&nbsp; je fluorescentni test u kvascu za identifikaciju potencijalnih prirodnih ili sintetičkih liganada&nbsp; ER&alpha;, ER&beta; ili AR i kvantifikaciju&nbsp; njihovog&nbsp; afiniteta&nbsp; vezivanja sa mogućno&scaron;ću testiranja čitavih biblioteka modifikovanih steroida i ksenoestrogena. Takođe, opisana&nbsp; je primena optimizovanog biosenzora&nbsp; za&nbsp; procenu&nbsp; estrogenog&nbsp; potencijala sintetskih steroida i odabranih biljnih ekstrakata bogatih jedinjenjima fitoestrogenih osobina. U cilju potpunijeg sagledavanja mehanizma&nbsp; delovanja&nbsp; odabranih&nbsp; modifikovanih&nbsp; steroida&nbsp; ispitana&nbsp; je&nbsp; njihova antiproliferativna aktivnost prema ćelijskim&nbsp; linijama estrogen receptor pozitivnog kancera dojke&nbsp; (MCF-7) i kancera prostate (PC-3), dok su&nbsp; in silico metodom molekularnog&nbsp; dokinga&nbsp; predviđene&nbsp; energije&nbsp; i&nbsp; geometrije&nbsp; vezivanja&nbsp; ovih&nbsp; jedinjenja za ligand-vezujuće&nbsp; domene&nbsp; ER&alpha; i ER&beta;. Drugi deo ovog rada obuhvata razvoj testa za&nbsp; ispitivanje aktivnosti humanog enzima aromataze,&nbsp; heterologno eksprimiranog u ćelijama&nbsp; kvasca&nbsp; Saccharomyces cerevisiae&nbsp; i/ili&nbsp; bakterija Escherichia coli, u prisustvu&nbsp; ili&nbsp; odsustvu&nbsp; inhibitora.&nbsp; Interakcije modifikovanih&nbsp; steroida, odabranih na osnovu strukture,&nbsp; sa&nbsp; aromatazom&nbsp; ispitane&nbsp; su&nbsp; osetljivim spektroskopskim metodama, praćenjem promene spinskog stanja Fe iz hem grupe ili promene fluorescencije ostatka&nbsp; triptofana&nbsp; iz&nbsp; aktivnog&nbsp; centra usled konformacione&nbsp; promene&nbsp; proteina, izazvane interakcijom sa ligandom. Razvijeni in vitro testovi bez upotrebe radioaktivnih izotopa su, osim&nbsp; visoke efikasnosti&nbsp; i&nbsp; bezbednosti&nbsp; po&nbsp; korisnika&nbsp; i&nbsp; okolinu, pokazali&nbsp; specifičnost&nbsp; i&nbsp; ekonomičnost&nbsp; u preliminarnom&nbsp; skriningu&nbsp; liganada&nbsp; steroidnih receptora&nbsp; i inhibitora aromataze. Jedinjenja&nbsp; kod kojih je detektovana&nbsp; značajna biolo&scaron;ka aktivnost mogu potencijalno poslužiti kao osnova za razvoj terapeutika u lečenju hormon-zavisnih bolesti i stanja, koja danas predstavljaju globalni zdravstveni problem.</p> / <p>In&nbsp; this&nbsp; doctoral&nbsp; dissertation,&nbsp; a&nbsp; fluorescent&nbsp; assay&nbsp; in&nbsp; yeast&nbsp; was&nbsp; developed&nbsp; for&nbsp; identification&nbsp; of&nbsp; potential&nbsp; natural or synthetic ligands of ER&alpha;, ER&beta; or AR and<br />quantification&nbsp; of&nbsp; their&nbsp; binding&nbsp; affinity,&nbsp; as&nbsp; well&nbsp; asevaluation&nbsp; of&nbsp; the&nbsp; estrogenic&nbsp; potential&nbsp; of&nbsp; synthetic steroids&nbsp; and&nbsp; selected&nbsp; plant&nbsp; extracts&nbsp; rich&nbsp; in phytoestrogen&nbsp; content.&nbsp; The&nbsp; assay&nbsp; could&nbsp; be&nbsp; used&nbsp; to&nbsp; screen&nbsp; libraries&nbsp; of&nbsp; modified&nbsp; steroids&nbsp; and xenoestrogens.&nbsp; In&nbsp; order&nbsp; to&nbsp; better&nbsp; understand&nbsp; the biomedical&nbsp; potential&nbsp; of&nbsp; selected&nbsp; modified&nbsp; steroids, results&nbsp; were&nbsp; compared&nbsp; to&nbsp; antiproliferative&nbsp; activity against&nbsp; estrogen&nbsp; receptor&nbsp; positive&nbsp; breast&nbsp; cancer (MCF-7)&nbsp; and&nbsp; prostate&nbsp; cancer&nbsp; (PC-3)&nbsp; cell&nbsp; lines. Binding&nbsp; energies&nbsp; and&nbsp; the&nbsp; geometry&nbsp; of&nbsp; binding&nbsp; of these&nbsp; compounds&nbsp; for&nbsp; ER&alpha;&nbsp; and&nbsp; ER&beta;&nbsp; ligand&nbsp; binding domains&nbsp; were&nbsp; predicted&nbsp; in&nbsp; silico&nbsp; by&nbsp; molecular&nbsp; docking&nbsp; methods.&nbsp; The&nbsp; second&nbsp; part&nbsp; of&nbsp; this&nbsp; study includes development&nbsp; of&nbsp; an&nbsp; assay&nbsp; for&nbsp; study&nbsp; of&nbsp; aromatase&nbsp; activity&nbsp; in&nbsp; the&nbsp; presence&nbsp; or&nbsp; absence&nbsp; of inhibitors&nbsp; by&nbsp; heterologous&nbsp; expression&nbsp; of&nbsp; human aromatase&nbsp; in&nbsp; Saccharomyces&nbsp; cerevisiae&nbsp; and/or Escherichia&nbsp; coli&nbsp; cells,&nbsp; as&nbsp; model-organisms. Furthermore, interactions between modified steroids, selected&nbsp; according&nbsp; to&nbsp; their&nbsp; structure,&nbsp; and&nbsp; aromatase were&nbsp; tested&nbsp; using&nbsp; sensitive&nbsp; spectroscopic&nbsp; methods based on ligand-induced changes&nbsp; in&nbsp; the&nbsp; spin state of Fe&nbsp; from&nbsp; the&nbsp; heme&nbsp; group&nbsp; or&nbsp; changes&nbsp; in&nbsp; the fluorescence&nbsp; of&nbsp; a&nbsp; tryptophan&nbsp; residue&nbsp; in&nbsp; the&nbsp; active site.&nbsp; The&nbsp; non-radioactive&nbsp; in&nbsp; vitro&nbsp; assays&nbsp; developed&nbsp; here, besides high efficiency, user and environmental safety,&nbsp; also&nbsp; have&nbsp; greater&nbsp; specificity&nbsp; and&nbsp; are&nbsp; more cost-effective&nbsp; for&nbsp; preliminary&nbsp; screening&nbsp; of&nbsp; steroid receptor&nbsp; ligands&nbsp; and&nbsp; aromatase&nbsp; inhibitors. Additionally,&nbsp; compounds&nbsp; identified&nbsp; to&nbsp; express significant biological activity can serve as a basis for the&nbsp; development&nbsp; of&nbsp; potential&nbsp; therapeutics&nbsp; in&nbsp; the treatment&nbsp; of&nbsp; hormone-dependent&nbsp; diseases&nbsp; and conditions, a global health issue today.</p>

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