In the recent years, environmental pollution becomes a health threatening issue for human beings. Technological developments introduce industrial wastes and heavy metals, and developments in agriculture introduce pesticides into the world that we live. All these toxic wastes accumulate in drinking water and food consumed by humans. Therefore, detection of toxic wastes in all kinds of environmental samples, and development of new detection techniques become an important issue. In this study, development of a protein-based biosensor for detection of heavy metals in environmental samples, by expressing genetically modified glutathione S-transferase (GST-(His)6) protein in E.Coli BL21 (DE3) expression system, was designed. Recombinant GST proteins was expressed in E.Coli BL21 (DE3) expression system and purified with Glutathione Sepharose 4B affinity column and Ni-NTA spin kit. GST activities were determined using the GST substrate 1-chloro-2,4-dinitrobenzene (CDNB). Protein expression was tested by SDS-PAGE and Western blot analysis. Product formation linearly increased up to 1 mM CDNB, 1 mM GSH, 1.7 &micro / g proteins in 0.05 M, pH 6.9 phosphate buffer in the final volume of 1.0 ml at 25&amp / #9702 / C. The Vmax and Km values for GST-(His)6 towards CDNB and GSH were calculated with Lineweaver-Burk as CDNB Vmax / 22.88 &micro / mol/min/mg, Km / 4.29 mM,and as GSH Vmax / 6.42 &micro / mol/min/mg, 24.45 &micro / mol/min/mg, Km / 3.69 mM, respectively. Biosensor working electrode was prepared by immobilizing the GST-(His)6 by 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC) coupling on the gold surface. Electrode preparation was confirmed by cyclic voltammetry measurements. The biosensor was inserted as the working electrode in the constructed three(four)-electrode flow cell. The conformational change resulting from the binding of the metal ions to the recombinant protein causing a capacitance change proportional to the concentration of the metal ions was determined. After the working electrode is standardized and calibrated, the heavy metal concentration in water samples was measured. The GST-(His)6 biosensor has a large operational range between 1 fM and 10 mM and a storage stability of approximately 2 weeks. The GST-(His)6 biosensor is very sensitive to, Cu+2&gt / Cd+2&gt / Zn+2&gt / Hg+2 metal ions, at low concentrations.

化学シナプスにおいて、シナプス小胞内に蓄えられた神経伝達物質の開口放出によってシナプス伝達が起こる。開口放出後、シナプス小胞はエンドサイトーシスによって細胞内へ回収されて再利用される。膜容量測定法とpHイメージングを併用して、開口放出とエンドサイトーシスに伴う小胞膜および小胞タンパク質の動態を同時測定した。Ca2+-カルモジュリン-Munc13シグナリングが小胞タンパク質の回収に関与していることが示唆された。 / Neurotransmitter is released at synapses by fusion of synaptic vesicles with the plasma membrane. To sustain synaptic transmission, membranes and vesicular proteins has to be retrieved for reuse. I have combined capacitance measurements and pH-imaging via a pH-sensitive vesicular protein marker, and compared the retrieval kinetics of membranes and synaptotagmin2 (Syt2) at the calyx of Held presynaptic terminal. Data in this thesis identifies a novel mechanism of stimulus- and Ca2+-dependent regulation of coordinated endocytosis of synaptic membranes and Syt2. / 博士(理学) / Doctor of Philosophy in Science / 同志社大学 / Doshisha University

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Hardware Security Modules (HSMs) are embedded systems that provide a physically secure data storage and handling environment. This master thesis evaluates an HSM method incorporating cryptographic key generation, key management, and tamper protection. The HSM concept involves a sensing mesh structured Physical Unclonable Function (PUF), where the cryptographic key is derived from the sum of cross-sectional area capacitance between conductors on adjacent layers of a flex PCB forming a grid. This sensing mesh PUF that stores a digital fingerprint in its microstructure is used to enclose an internal system extracting and managing the keys. This ensures that accessing the internal structure is unmanageable without modifying the enclosure. Since the cryptographic key is derived from the intrinsic properties within the sensing mesh, modifying it will change its intrinsic properties and change the cryptographic key and make it unusable. The Master thesis contains PCB design and development of a prototype of the PUF system and an associated capacitance measurement system, which can handle and extract unique keys from each copy of the PUFs. A hardware assembling, experimenting, and evaluation procedure were performed regarding the robustness of the PUF and its susceptibility to environmental impacts such as temperature changes, invasive attacks, and agitation. Additionally, an performance evaluation is made by estimating a set of quality factors often associated with PUFs, such as uniqueness, reliability, uniformity, and bit-aliasing on the extracted cryptographic keys. The cryptographic keys provide good reliability in stable conditions for each PUF copy of the population. The cryptographic keys also provide gooduniqueness, uniformity, and bit-aliasing estimations with the quality factors. Moreover, an invasive attack experiment indicates that the PUF enclosure prototype provides tamper detection possibilities together with distinct structure modifications when an intrusion attempt is performed. As stated in theory, PUFs are sensitive to environmental changes, which is also observable in the results when the PUF enclosure prototype is exposed to various environmental conditions. / <p>Examensarbetet är utfört vid Institutionen för teknik och naturvetenskap (ITN) vid Tekniska fakulteten, Linköpings universitet</p>

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