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Large-Scale Kinetic Analyses of Protein-Protein Interactions: Advancing the Understanding of Post Translational Modifications in Biological RegulationJanuary 2018 (has links)
abstract: Signal transduction networks comprising protein-protein interactions (PPIs) mediate homeostatic, diseased, and therapeutic cellular responses. Mapping these networks has primarily focused on identifying interactors, but less is known about the interaction affinity, rates of interaction or their regulation. To better understand the extent of the annotated human interactome, I first examined > 2500 protein interactions within the B cell receptor (BCR) signaling pathway using a current, cutting-edge bioluminescence-based platform called “NanoBRET” that is capable of analyzing transient and stable interactions in high throughput. Eighty-three percent (83%) of the detected interactions have not been previously reported, indicating that much of the BCR pathway is still unexplored. Unfortunately, NanoBRET, as with all other high throughput methods, cannot determine binding kinetics or affinities. To address this shortcoming, I developed a hybrid platform that characterizes > 400 PPIs quantitatively and simultaneously in < 1 hour by combining the high throughput and flexible nature of nucleic programmable protein arrays (NAPPA) with the quantitative abilities of surface plasmon resonance imaging (SPRi). NAPPA-SPRi was then used to study the kinetics and affinities of > 12,000 PPIs in the BCR signaling pathway, revealing unique kinetic mechanisms that are employed by proteins, phosphorylation and activation states to regulate PPIs. In one example, activation of the GTPase RAC1 with nonhydrolyzable GTP-γS minimally affected its binding affinities with phosphorylated proteins but increased, on average, its on- and off-rates by 4 orders of magnitude for one-third of its interactions. In contrast, this phenomenon occurred with virtually all unphosphorylated proteins. The majority of the interactions (85%) were novel, sharing 40% of the same interactions as NanoBRET as well as detecting 55% more interactions than NanoBRET. In addition, I further validated four novel interactions identified by NAPPA-SPRi using SDS-PAGE migration and Western blot analyses. In one case, we have the first evidence of a direct enzyme-substrate interaction between two well-known proto-oncogenes that are abnormally regulated in > 30% of cancers, PI3K and MYC. Herein, PI3K is demonstrated to phosphorylate MYC at serine 62, a phosphosite that increases the stability of MYC. This study provides valuable insight into how PPIs, phosphorylation, and GTPase activation regulate the BCR signal transduction pathway. In addition, these methods could be applied toward understanding other signaling pathways, pathogen-host interactions, and the effect of protein mutations on protein interactions. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2018
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Fitohemijski sastav, lekoviti potencijal i senzorne karakteristike sorti vrsta Brassica oleracea L. i Brassica rapa L. (Brassicaceae) iz organskog i konvencionalnog sistema proizvodnje / The phytochemical composition, healing potential and sensory characteristics of cultivars of Brassica oleracea L. i Brassica rapa L. species (Brassicaceae) from organic and conventional cropping systemsBošković Svetlana 29 September 2016 (has links)
<p>U ovom radu su ispitani hemijski sastav i biološka aktivnost vrsta <em> Brassica oleracea </em>i<em> Brassica rapa</em> poreklom iz organskog i konvencionalnog sistema proizvodnje. Ispitivanja hemijskog sastava su obuhvatila određivanje volatilnih komponenata primenom headspace GC-MS tehnike, kvalitativnu analizu sokova ispitivanih vrsta pomoću LC-DAD-MS/MS tehnike, kvantifikaciju odabranih fenolnih jedinjenja pomoću LC-MS/MS tehnike i spektrofotometrijsko određivanje sadržaja ukupnih glukozinolata, fenola i flavonoida i sadržaja ukupnih monomernih antocijana. Biološka aktivnost povrća je ispitana određivanjem antioksidantnog potencijal, antimikrobne i antitumorske aktivnosti sokova. Dobijeni rezultati pokazuju da su glavne volatilne komponente ispitivanih vrsta degradacioni proizvodi masnih kiselina i organosumpornih jedinjenja od kojih neke od identifikovanih komponenata predstavljaju ključne mirisne komponente. Na osnovu njihove distribucije nije bilo moguće utvrditi uticaj sistema proizvodnje na miris i ukus ispitivanih vrsta. Pomoću LC-DAD-MS/MS i LC-MS/MS tehnike je utvrđeno prisustvo primarnih metabolita, slobodnih hidroksibenzoevih i hidroksicimetnih kiselina, derivate hinske kiseline sa hidroksicimetnim kiselinama, glikozida flavonola, biflavonoida, i kumarina u sokovima spitivanih vrsta. Pored toga u svim sokovima subili prisutni kondenzacioni proizvodi degradacionih proizvoda indolnih glukozinolata sa askorbinskom kiselinom- askorbigen i metoksiaskorbigen. U sokovima crvenog kupusa su bili prisutni antocijani. Najveći sadržaj ukupnih glukozinolata je određen u brokoliju, dok je crveni kupus sadržao najveći sadržaj ukupnih fenola ukupnih flavonoida kao i hinske kiseline. Crveni kupus je bio varijetet koji je ispoljio najviši antioksidantni kapacitet u svim primenjenim testovima, brokoli i karfiol su ispoljili najjaču antimikrobnu aktivnost, dok je kelj bio varijetet sa najjačim antitumorskim delovanjem. Na osnovu analize hemijskog sastava i biološke aktivnosti vrsta <em>B</em>. <span id="cke_bm_177S" style="display: none;"> </span><em>oleracea</em><span id="cke_bm_177E" style="display: none;"> </span> i <em> B. rapa</em> može se <span id="cke_bm_178E" style="display: none;"> </span>zaključiti da u zavisnosti od primenjenog tipa proizvodnje mogu biti poboljšane neke od karakteristika ispitivanih vrsta i da je uticaj sistema proizvodnje značajan za pojedine varijetete, a nikako za celu vrstu. U konačnom obliku može se zaključiti da bi potencijalna kombinacija sokova crvenog kupusa, brokolija, karfiola i kelja mogla ostvariti značajno hemopreventivno i hemoterapeutsko delovanje, ispoljavajući istovremeno antioksidantnu, antimikrobnu i antitumorsku aktivnost.</p> / <p>Chemical composition and biological activity of<em> Brassica oleracea</em> and <em>B. rapa </em> species were investigated in the present work. Investigation of chemical composition encompassed determination of volatile compounds by headspace GC-MS technique, qualitative analysis of juices of investigated vegetables by LC-DAD-MS/MS technique, quantification of selected phenolic compounds by LC-MS/MS technique and spectrophotometric determination of total content of glucosinolates, phenolic compounds and flavonoids and total content of monomeric anthocyanes. Biological activity of vegetables was investigated by determination of antioxidant capacity, antimicrobial and antitumorigenic effect of juices. Obtained results showed that main volatile compounds were degradation products of fatty acids and organo-sulphur compounds among which some of the identified components were the key aroma compounds. Based on their distribution it was not possible to evaluate influence of cropping system on aroma and taste of investigated species. By LC-DAD-MS/MS and LC-MS/MS techniques, it was determined the presence of primary metabolites, freehydroxybenzoic and hydroxycinnamic acids, derivatives of quinic acid and hidroxicinnamic acids, flavonol glucosides, biflavnoids and a cumarin in juices of the investigated species. Furthermore, condensation products of degradation products of indole glucosinolates with ascorbic acid- ascorbigen and metoxiascorbigen were present in all juices. Anthocyanes were present in juices of red cabbage. The highest total content of glucosinolates was determined in broccoli, while red cabbage had the highest total phenolic content, total flavonoid content, as well as content of qunic acid. Red cabbage was a variety that showed the highest antioxidant capacity measured by different assays. Broccoli and cauliflower expressed the highest antimicrobial potential, while Savoy cabbage was a variety with the highest antitumor effect. Investigation of chemical composition and biological activity showed that applied cropping conditions may be useful for improving some of the characteristics of investigated species and significance of cropping system only for particular varieties, but not for the whole species in general. As the conclusion, potential combination of juices of red cabbage, broccoli, cauliflower and Savoy cabbage could be potent chemopreventive and chemotherapeutic agent, expressing simultaneously antioxidant, antimicrobial and antitumor activity.</p>
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