Magnetic nanoparticles have been used in many areas owing to their variable characteristic behaviors. Among these iron oxide nanoparticles are one of the mostly preferred type of nanoparticles. In this study Fe3O4, namely magnetite, which is one type of magnetic iron oxide nanoparticles was used. Magnetite nanoparticles with a narrow size distribution were prepared in aqueous solution using the controlled coprecipitation method. They were characterized by electron microscopic methods (SEM and TEM), crystal structure analysis (XRD), particle size analyzer, vibrating sample magnetometer (VSM) and Raman spectrometry. The nanoparticles were coated with a thin (ca 20 nm) silica shell utilizing the hydrolysis and the polycondensation of tetraethoxysilane (TEOS) under alkaline conditions in ethanol. The presence of silica coating was investigated by energy dispersive X-ray spectrometer (EDX) measurement. After surface modification with an amino silane coupling agent, (3-Aminopropyl)triethoxysilane, histidine was covalently linked to the amine group using glutaraldehyde as cross-linker. Carbonyl complexes of rhenium [Re(CO)3(H2O)3]+ was prepared through reductive carboxylation utilyzing gaseous carbon monoxide as a source of carbonyl and amine borane (BH3NH3) as the reducing agent. The complex formation was followed by HPLC- ICP-MS system and 95% conversion of perrhanete into the complex was achieved. The magnetic nanoparticles were then labeled with the Re complex with a yield of 86.8% through the replacement of labile H2O groups with imidazolyl groups. Thus prepared particles were showed good stability in vitro. Herein rhenium was selected as a surrogate of radioactive 99mTc. However radioactive isotopes of rhenium (186-Re and 188 Re) is also used for radioactive therapy.

MPLS, a next generation backbone architecture, can speed up packet forwarding to destination by label switching. However, if there exists no backup LSP when the primary LSP fails, MPLS frames cannot be forwarded to destination. Therefore, fault recovery has become an important research area in MPLS Traffic Engineering. Makam approach and Haskin approach are the most famous two among the previous literatures. Besides, IETF has made strict definitions for MPLS Recovery in RFC 3469 in February, 2003. We propose a Reverse Labeling Scheme to handle fault recovery in this thesis. We establish a virtual reverse LSP along the completely reverse direction of the primary path. When there is a link failure in the primary LSP, LSR will forward packets back to Ingress by virtual reverse LSP instead of using the primary LSP. This idea of building virtual reverse LSP makes Haskin approach practical in implementation. In addition, we save network resources by designing a scheme such that LSR is easier to convert from the primary LSP to the backup LSP. In order to solve the out-of-order packets in Haskin approach, Hundessa adds buffering on every LSR. The buffer can temporarily store the packets once a link failure has been detected. By adopting the basic idea of Hundessa approach, we embed our Reverse Labeling Scheme and implement it on Linux platform. We also make some modifications to solve the buffering problems. Finally, we demonstrate this Reverse Labeling Scheme by several experiments. We not only show the low packet loss rate, but also solve the packet out-of-order problems. The significant decrease of out-of-order packets can further improve the efficiency of TCP flow transmission.

The structure and function of RNA molecules are dependent on RNA-metal ion interactions in both diffusive and direct ways. Structural information for RNA has been obtained using various biophysical and biochemical methods. In this study, using site-directed spin labeling (SDSL) and EPR spectroscopy, distances in RNA duplexes, TAR RNA, and the hammerhead ribozyme have been measured to investigate RNA structures. Kinetic measurements have been performed in the extended hammerhead ribozyme to correlate the catalytic function with metal dependent ribozyme folding. As a basic model system for distance measurements, inter-spin distances in RNA duplexes with spin labels at various positions are measured using SDSL with continuous EPR and a Fourier deconvolution method. Divalent metal-ion dependent TAR RNA folding from bent to extended conformers is monitored by measuring inter-spin distances near the bulge region. In order to investigate a proposed loop-loop interaction in the extended hammerhead ribozyme which significantly enhances the ribozyme activity, distance measurements, dynamics studies, and kinetics measurements have been performed. We have introduced PELDOR long-distance measurements in order to investigate metal dependent folding of the hammerhead ribozyme. The dynamics of the spin labels attached to the hammerhead ribozyme with increasing mono- and divalent metal ion concentrations are monitored using CW EPR spectroscopy at room temperature. EPR data show that a loop-loop interaction occurs near the U1.6 nucleotide, and that in 0.1 M NaCl the docking occurs at submillimolar Mg2+ concentrations ([Mg2+]1/2, docking = ~ 0.7 mM). Kinetics measurements show that the hammerhead ribozyme requires high concentration of Mg2+ for the maximum cleavage activity ([Mg2+]1/2, cleavage = ~ 90 mM).

Proteins have evolved over millions of years to serve a plethora of highly specialized functions in biological systems. Given the enormous diversity in structure and function, it is truly surprising that only 20 different amino acids are utilized as the building blocks of proteins. Furthermore, only a small set of metal cations that are biologically available are used as structural or catalytically active cofactors in proteins, whereas rare metal cations such as platinum, ruthenium or rhodium remain absent. In the 20th century myriad catalysts, based on non-biological transition metals, emerged that can facilitate numerous organic transformations. The goal of the thesis was to introduce new functions into proteins by attaching platinum metals and fluorescent metal sensors. Thus, semi-synthetic proteins for catalytic and analytical applications were generated. The replacement of organic solvents by environmentally benign solvents such as water is an imperative step towards achieving "green chemistry". The combination of small molecule catalysts with proteins may introduce new functions and take advantage of the benefits of "both worlds" while avoiding their potential drawbacks. Therefore semi-synthetic catalysts were developed for enantioselective organic reactions in aqueous medium. A suitable reaction, reaction conditions and catalytic system for later utilization in a semi-synthetic protein were designed, developed and characterized. Ruthenium porphyrins catalyzed cyclopropanation reactions with fair yields and high stereoselectivity in aqueous medium. The successful reaction in water was a crucial requirement for a catalytically active semi-synthetic protein. Mechanistic studies did not elucidate the actual catalytic species for the formation of the cyclopropanation product and the side-product diethyl maleate; however, new insights were gained from the analysis of potential reaction pathways. Moreover, studies of the influence of axial ligands, resembling likely residues coordinating to the ruthenium metal center in the active site of a semi-synthetic protein, on the carbene formation of ruthenium porphyrins illustrated that coordination of axial ligands may inhibit the catalytic activity. The generation of ruthenium porphyrin based semi-synthetic proteins and their subsequent catalysis of cyclopropanation reactions was carried out. Myoglobin and myoglobin mutants were successfully reconstituted with a heme-like ruthenium carbonyl porphyrin; however, none of the formed semi-synthetic proteins catalyzed the enantioselective cyclopropanation of styrene. Efforts to determine the reconstitution efficiency of the generated semi-synthetic were hampered by problems to purify the generated semi-synthetic proteins that are probably due to non-specific binding of the ruthenium porphyrin to the protein surface. The exploration of labile metal pools of the biologically relevant transition metals copper, iron and zinc in cells was the goal of developing semi-synthetic proteins for analytical applications. Combining fluorescent proteins with colored or fluorescent metal chelators by forming semi-synthetic proteins allows taking advantage of their beneficial properties while avoiding their downsides. This design offers an attractive platform for in vivo metal sensing. Plasmids encoding fluorescent proteins, targeting sequences and AGT or intein fusion domains (necessary for labeling) for eukaryotic and prokaryotic expression were generated. The targeting of intracellular compartments (mitochondria, nucleus and TGN) was successful (confirmed by light microscopy experiments with transfected mammalian cells). In vitro labeling experiments of expressed and purified fusion proteins with rhodamine derivatives succeeded with AGT based fusion proteins; however, labeling of fusion proteins by trans-splicing with split-inteins failed. A new Zinc(II)-chelator was attached to an AGT based protein and the resulting semi-synthetic protein exhibited strong changes of fluorescence in the presence of zinc(II). This represents an important step towards the goal of in vivo cell imaging of labile zinc(II) pools. Despite extensive efforts, all attempts failed to generate a chelator that forms Cu(I)-complexes with the 1:1 stochiometry (ligand:metal) that is necessary for metal sensing with semi-synthetic proteins.

published_or_final_version / Public Health / Master / Master of Public Health

Eukaryotic genomes are densely compacted into chromatin, so that they can be contained in the nucleus. Despite the tight packaging genes need to be accessible for normal metabolic activities to occur, such as transcription, repair and replication. These processes are regulated by a vast number of proteins but also by the level of compaction of chromatin. The translocation of motor proteins along DNA produces torsional stress which in turn alters chromatin compaction both upstream and downstream. Few single-molecule studies have investigated the behaviour of nucleosomes when subjected to torsion. The inability to measure the applied torque though represented a major limitation to those reports. The implementation of the rotor bead assay, which allows to directly measure the torque applied in magnetic tweezers experiments, has been hindered by a difficult sample preparation procedure. In order to overcome this limitation an efficient protocol for the insertion of chemical or structural modifications in long DNA substrates was developed. This was then further expanded to allow the introduction of labels in multiple loci and/or both strands and has been used successfully in a number of studies. Furthermore this is the first report of tensile experiments performed on nucleosomes with a histone variant. H2AvD nucleosomes were studied due to the interest in the biological role of H2A.Z-family proteins. Interestingly, the variant nucleosomes appear to bind less DNA and to be evicted from the DNA at lower forces than those observed for canonical nucleosomes. These findings show an important role for the H2A-H2B dimers in the mechanical stability of nucleosomes. Furthermore these results are in agreement with recently proposed models of a dynamic nucleosome, in contrast to the long-standing view of nucleosomes as static structures.

No description available.

Phospholipid vesicles are biocompatible, and have potential for intracellular applications, but minimal membrane integrity limits their use in membrane-rich environments. Stabilized membranes overcome this limitation while maintaining biocompatible surface structures. Additionally, the modularity of phospholipid bilayer makes them ideal components when designing biologically inspired sensors. Membrane composition can be tailored to specific applications, transmembrane proteins can provide added functionalities, and the isolated interior can prevent cytotoxic and interfering detection chemistries from altering the cellular environment. This work has focused on expanding the capabilities of stabilized phospholipid membranes, and determining which formulations hold promise in developing stabilized phospholipid vesicle nanosensors.Current membrane stabilization methods suffer from either incomplete stabilization, or irreversible stabilization limiting the applications of vesicle nanosensors. Therefore, a facile method to prepare robust phospholipid vesicles using commonly available phospholipids stabilized via the formation of an interpenetrating, acid-labile, cross-linked polymer network that imparts controlled polymer destabilization and subsequent vesicle degradation was developed. Upon exposure to acidic conditions, the highly cross-linked polymer network was converted to linear polymers, substantially reducing vesicle stability upon exposure to chemical and physical insults. The resultant transiently stabilized vesicles have potential for enhanced drug delivery and chemical sensing applications requiring minimal membrane defects, and allow for improved physiological clearance.Some vesicle nanosensor schemes may require the passive diffusion of low molecular weight species across the membrane in addition to controllable degradation. Therefore, the acid-degradable, polymer-stabilized, phospholipid vesicle production method was extended to bis-SorbPC membranes by simultaneously polymerizing the vesicle with an acetal-containing cross-linker. The vesicles display prolonged stability under physiological conditions, and significant additional stability compared to vesicles composed of naturally occurring phospholipids. The vesicles demonstrated potential utility for sensing and therapeutic applications.Phospholipid vesicles can also serve as labels to observe movement in macromolecular biological assemblies, but a dearth of caged fluorescent labels limits design and function. Therefore, the first caged fluorescent thiol was synthesized, shown to label amines rapidly, and demonstrated the required photolytic properties. The caged fluorescent thiol has potential as a label in observing the movement of macromolecular biological assemblies and as a fluorescent probe for observing endosomal trafficking and release.

Research Question/Purpose: Due to environmental legislation, economic influences and increasing concern about the environment among the general public, today’s businesses are becoming more committed to environmental issues. Some enterprises yet have implemented a green strategy. This thesis aims at identifying issues that determine the long-term efficiency of green marketing and how confidence and trust plays a role in order to gain competitive advantage through customer satisfaction and customer retention. The common denominator of both, CRM and green marketing is the creation of confidence, trust and value for customers. So, focus is laid on measures that lend credibility to companies’ green marketing by interviewing experts and observing their opinions about green marketing and eco-labeling. Design/Methodology/Approach: Interviews conducted with company professionals from IKEA and Konsum Gävleborg and a member of the municipality in Gävle, as well as findings from secondary sources of the company Nestlé were used to investigate the companies’ ways to respond to environmental concern and how they deal with green issues and ecological responsibility. The interviews provide valuable insights of the success of green marketing depending on the confidence between company and customer. Findings: By analyzing the results of the interviews with the literature review, the thesis reveals that environmental responsibility and ecological orientation needs to be based upon the entire business strategy of a company and implemented in its core principles in order to deal effectively and efficiently with this issue. Conclusions: Companies respond in different ways to ecological issues, through eco-labeling, cooperation with NGOs and energy-saving production processes. In conclusion, it can be said that the need for customer relationship commitment, trust, confidence and loyalty as a result of satisfaction are as important in customer relationship management as in green marketing, because competitive advantage can be achieved by environment-related activities and motivates companies to go green to promote ecologically sustainable practices. Research Limitations/Implications: There is a number of literature focusing on customer relationship management and implications of green issues for business strategy, but there are only a few on green marketing yet and the interaction between green marketing and CRM is missing attention, so far. Originality/Value: This work seeks to make a contribution towards bridging the ends of CRM and green marketing. It indicates a relationship between eco-orientation and company performance and implicates, on the basis of the theoretical and empirical findings, that integrating ecological features and good performance in a company is not impossible. Trust and confidence are as important concepts in green marketing as in CRM.

No description available.