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1	Luminescent Quantum dots for Cellular Analysis Shi, Lifang 15 December 2007 (has links) Luminescent quantum dots have attracted great interest in recent years among biological researchers since they provide solutions to problems associated with use of organic fluorophores in cellular studies. Quantum dots show high photostability, high emission quantum yield, narrow and symmetric emission peaks and size-dependent wavelength tunability. The objective of my PhD studies was to develop CdSe/ZnS quantum dot-based probes and utilize them in cellular assays. The first phase of the work was to develop luminescent quantum dot fluorescence resonance energy transfer (FRET) based probes for protease activity. The probes were based on FRET interactions between quantum dots that serve as donors and rhodamine molecular acceptors that were immobilized to the surface of the quantum dots through peptide linkers, which contained selective enzymatic cleavage sites. Upon enzymatic cleavage of the peptide linkers, the rhodamine molecules no longer provided an efficient energy transfer channel to the quantum dots, which brightened the previously quenched quantum dots. The probes were applied to detect enzyme activity, screen enzyme inhibitors, and discriminate between normal and cancerous cells primarily because of the difference in the proteolytic activity in extracellular matrices. The second phase of my work was to take advantage of FRET and quantum dots to develop pH sensor. First quantum dots were modified with metallothionein (MT) to be water-soluble and biocompatible. The MT-coated quantum dots were labeled with Rhodamine through the formation of amide bonds with å-amine group of lysine in MT peptide to form the probes. FRET efficiency between quantum dots (donor) and rhodamine (acceptor) was pH dependent. The final phase of my studies focused on the first preparation of reversible quantum dot-based cellular probes for labile iron. The MT coated quantum dots was modified with EDTA to form probes. When captured by the EDTA molecules, iron ions quenched the emission of quantum dots. Removal of iron from the quantum dot surface by free EDTA or other iron chelators with higher binding affinity resulted in a rise in the luminescence of quantum dots. The analytical properties of the probes including sensitivity, selectivity, and reversibility were characterized. Intracellular assays in iron-enriched astrocytes will be carried out. Quantum dots FRET Protease sensor pH sensor Iron sensor Cellular analysis
2	Purification, functional characterization and crystallization of the PerR peroxide sensor from Saccharopolyspora erythraea Elison Kalman, Grim January 2019 (has links) This report summarizes the work on the cloning, expression, and purification of PerR, a metal sensing regulator from Saccharopolyspora erythraea and the subsequent characterization using small angle X-ray scattering and other biochemical methods. The report aims to provide an insight into prokaryotic metal homeostasis, provide a better understanding of how PerR works and provide valuable information for the continued work on the crystallization of PerR. structural biology cell culture protein expression genetic engineering spectroscopy X-ray crystallography small angle X-ray scattering SAXS transcription factor PerR metal binding antibiotics Saccharopolyspora erythraea S. erythraea soil bacterium actinobacteria actinomycetes oxidative stress peroxide stress structure determination characterization stabilization peroxide sensor iron sensor manganese sensor metalloprotein metal ion homeostasis metal ion prokaryotic strukturbiologi cellodling proteinuttryck genteknik spektroskopi röntgenkristallografi småvinkel-röntgenspridning SAXS transkriptionsfaktor PerR metallbindande antibiotika Saccharopolyspora erythraea S. erythraea jordartsbakterie Aktinobakterier Actinobacteria Actinomycetes oxidativ stress peroxid stress strukturbestämning karaktärisering stabilisering peroxidsensor järnsensor mangansensor metalljonshomeostas metalljoner prokaryot Structural Biology Strukturbiologi

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Luminescent Quantum dots for Cellular Analysis

Purification, functional characterization and crystallization of the PerR peroxide sensor from Saccharopolyspora erythraea