Zinc is an essential metal ion for the activity of multiple enzymes and transcription factors. Among many other transporting proteins human serum albumin (HSA) is the main carrier of Zn(II) in the blood plasma. HSA displays multiple ligand binding sites with extraordinary binding capacity for a wide range of ions and molecules including fatty acids. Hence, HSA controls the availability and distribution of those molecules throughout the body. Previous studies have established that the existence of one zinc site with high affinity (MBS-A) that is modulated by the presence of fatty acids. Therefore, the fatty acid concentration in the blood influences zinc distribution which may result in a significant effect on both normal physiological processes and a range of diseases. Based on the current knowledge of HSA's structure and its coordination chemistry with zinc ion, here, we attempted to investigate zinc interactions and coordination with HSA and the effect of different fatty acids on the protein structure, stability and on Zn(II) binding. By NMR titration, we examine the Zn(II) binding to HSA and the spectra show distinct movements of some resonances showing a conformational change has occurred as a result of Zn(II) binding. Isothermal calorimetry titrations study was performed to evaluate zinc binding affinity to HSA in the absence and presence of fatty acids. Free HSA results indicates the existence of one high affinity site and multiple low affinity sites. Upon the binding of fatty acids to HSA, three distinct behaviors of Zn(II) affinity was observed ranging from no effect to moderate to significant depending on the FAs. By the use of circular dichroism, we investigate secondary and tertiary structure of HSA in the presence and absence of FAs and Zn(II). We found albumin is predominately α-helical and the overall conformation of the protein remains unchanged even after interacting with FAs and Zn(II) with some exception. The structural stability of HSA was evaluated by obtaining the denaturation temperature in the presence and absence of fatty acid and we found the thermal denaturation of HSA increases with the increase of amount of fatty acids.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18898号 / 医博第4009号 / 新制||医||1009(附属図書館) / 31849 / 京都大学大学院医学研究科医学専攻 / (主査)教授 木原 正博, 教授 中川 一路, 教授 一山 智 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Final publication is available at: http://journal.frontiersin.org/article/10.3389/fmicb.2015.00270/full / 付記する学位プログラム名：グローバル生存学大学院連携プログラム / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19619号 / 医博第4126号 / 新制||医||1015(附属図書館) / 32655 / 京都大学大学院医学研究科医学専攻 / (主査)教授 中川 一路, 教授 木原 正博, 教授 松林 公蔵 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

This is an exploration of the potential for a citizen science project, with the goal to get the general public involved in microbial soil biodiversity around Uppsala, Sweden. Biodiversity serves an important role in how an ecosystem performs and functions. A large part of Earth's biodiversity exists below ground in soil, where microorganisms interact with plants. It would be beneficial to analyse the abundance and spread of some microorganisms in order to gain a better understanding of soil biodiversity. We suggest that one species family to study could be Phytophthora. Phytophthora is a genus of oomycetes that often are pathogenic, causing disease in various trees and other plants. It is unknown exactly how widespread the genus is today, making it extra interesting for the proposed study. For the general public to be able to do this a device needs to be developed that is easy to use and preferably could be used directly in the field. An isothermal amplification method is suitable for identifying the microorganism under these conditions. Many isothermal amplification methods are expensive, perhaps too expensive for a citizen science study, but have great potential for easy field testing. We propose a device utilizing RPA and lateral flow strips. RPA - Recombinase Polymerase Amplification is a method for amplification that might be suitable since it is simple, sensitive, and has a short run time. It is however expensive, which is an issue, but isothermal amplifications are expensive across the board. Lateral flow strips can be used to visualize the results. They utilize antibodies to detect the previously amplified amplicons, and give a positive or negative test answer that would be understandable to even untrained study participants. One of the biggest obstacles identified in this project concerns amplifying DNA from a soil sample, because an extraction step is necessary. The methods we have identified for extraction are not performable in the field, since they require centrifugation. In the proposition for a device a possible work-around for this is proposed, but since it has yet to be tested it is not yet known whether it will work or not.

In this thesis, a calculation model is created to study a theoretical radiator-like configuration, consisting of a flat vertical plate heated with a constant capacity rate. This lumped capacitance model is partly created to more theoretically look at radiators with add-on-fans, but also to in such a setting look at fundamental heat transfer relationships. System heat transfer is studied for various heights, H (m), and freestream velocities, u (m/s). These results are then subject to validation, where comparison is made with values derived from two relevant reference studies. It is found that polynomial fits well describe the results obtained from calculation. The relationships for heat transfer Q (W), heat flux q (W/m2) thus become: 𝑄(𝐻,𝑢) = 𝑎00 + 𝑎01𝑢 + 𝑎10𝐻 + 𝑎11𝐻𝑢 + 𝑎02𝑢2 (W) 𝑞(𝐻,𝑢) =𝑄/𝐻= 𝑎00𝐻-1 + 𝑎01𝐻-1𝑢 + 𝑎10 + 𝑎11𝑢 + 𝑎02𝐻-1𝑢2 (W/m2) For these relationships, polynomial coefficients 𝑎00, 𝑎01, 𝑎10, 𝑎11 and 𝑎02 are found for three temperature set-ups of system supply and return temperature at zero freestream velocity: 55/45, 45/35 and 35/25 (°C). These values are chosen as they correspond to standard temperatures for low-temperature heating set-ups. Model validation is successful for the case of natural convection (u = 0), whereas difficulties are encountered for the cases of mixed and forced convection. Reasons for these difficulties are discussed and it is concluded that there is a need for more experimental studies of flat vertical plates with non-isothermal wall temperature profiles.

Calcineurin (CaN) plays an important role in T-cell activation, cardiac system development, and nervous system function. Previous studies have suggested that the regulatory domain (RD) of CaN binds Calmodulin (CaM) towards the N-terminal end of CaN. Calcium-loaded CaM activates the serine/threonine phosphatase activity of CaN by binding to the regulatory domain, although the mechanistic details of this interaction remain unclear. It is thought that CaM binding at the RD displaces the auto inhibitory domain (AID) from the active site of CaN, which activates phosphatase activity. In the absence of calcium-loaded CaM, the RD is at least partially disordered, and binding of CaM induces folding in the RD. Previous studies have shown that an ?-helical structure forms in the N-terminal half of the RD, but organization may occur in the C-terminal region as well. Here, we are presenting a model for the structural transition of the full length RD as it binds to CaM. Using nuclear magnetic resonance (NMR) spectroscopy, we have successfully assigned >85% of the 15N, 13C?, 13C? and HN chemical shifts of the unbound, regulatory domain of CaN. Secondary chemical shifts support a model where the RD is highly disordered. Our study of the CaM and CaN interaction supports the formation of a distal helix in the region between the AID and calmodulin-binding region. Heat capacity changes upon binding predict that 43 residues fold when CaM binds to CaN, consistent with the formation of this distal helix. Paramagnetic relaxation enhancement (PRE) studies of this interaction suggest a potential binding mode where the distal helix binds to CaM near residues I10-A11. Mutagenesis in the distal helix disrupts PREs, further supporting this hypothesis. Together, these data suggest that the interactions between CaM and the distal helix of CaN can be important in regulation of phosphatase activity.

No description available.

The dairy industry uses enzymes to make cheese, alter product flavor, and eliminate lactose. The activities of these enzymes have been measured in clear buffered solutions, but because of the limitations of spectrophotometric methods, enzyme activities have not been measured in opaque or colored dairy products where they are used. Isothermal titration calorimetry (ITC) can be used to determine reaction kinetics in opaque and colored solutions by measuring the heat rate from enzyme-catalyzed reactions as a function of time. This study used ITC to measure β-galactosidase activity in opaque solutions of milk, sweet whey, sweet whey permeate, acid whey, and acid whey permeate with two β-galactosidase (EC 3.2.1.23) isozymes derived from A. oryzae and K. lactis. The components of the dairy fluids alter the enzyme kinetics and reaction thermodynamics, and the reactions catalyzed by the two homologs differ as shown by differing thermodynamic profiles. The study demonstrates that ITC can be used to measure enzyme activity in opaque and colored dairy fluids and identify reactions by their thermodynamic properties. To ensure that ITCs are accurately recording heat data they must be calibrated regularly. However, potential problems have been identified with standard electrical calibration procedures; primarily being that the calibration is performed outside of the sample cell. This implies that any loss of heat from the theoretically adiabatic sample cell or loss of signal through led wires would be ignored by the electrical calibration. This research describes a new means for the chemical calibration of ITCs by performing acid-base titrations into the sample cell with KHP and TRIS base. This method for reaction was shown to be accurate to theoretical values across multiple temperatures and with different models of ITCs. Measurement errors due to diffusion of substrate are described along with means for limiting this factor. The method identified provides a procedure for maintaining the accuracy of ITCs by comparing their data to well-known thermodynamic values. It is anticipated that the simplicity and low-cost for running this calibration method will further standardize ITCs, help establish the ITC as a reliable method for measuring enzyme kinetics, and will make their maintenance simple enough for their use in quality assurance and industry settings.

Electronics, especially, printed circuit boards (PCBs) are a widespread technology. Metal coatings or “surface finishes” are often added to PCB board pads and component leads during manufacturing to improve their performance. Electroplated nickel/gold over copper is a popular surface finish for printed circuit boards and component leads. The presence of gold in solder joints, however, is known to have detrimental effects referred to as gold embrittlement. It is generally understood that tin-lead solder joints with less than 3 weight percent (wt%) of gold will not experience reliability issues. The acceptable level of gold in lead-free solder joints, however, is less well understood, as the technology is younger. The purpose of this study was to investigate the effect of gold content on the microstructural evolution of SnAgCu solder joints. Three integrated circuit packages with various thicknesses of gold coatings were assembled on boards that were made with thin (flash) or thick gold over nickel coatings. The boards were divided into three groups based on the isothermal aging they underwent: 0 days, 30 days, or 56 days of aging at 125 °C. Thirty four of the forty boards then underwent mechanical reliability testing. Components were cross-sectioned and polished. Scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS/EDX) were used to characterize the morphology and elemental composition of the solder joints and any intermetallic compounds (IMCs) that formed. The growth of bulk and interfacial layer IMCs in each package/board system was studied. In thick gold boards, AuSn4 particles observed in the bulk solder grew larger over time, absorbed Ni, and migrated to the component and board interfaces. (Cu1-p-qAupNiq)6Sn5 and (Au1-xNix)Sn4 IMCs were found at most board and component interfaces after aging. It was observed that most fractures occurred in or along the (Au1-xNix)Sn4 IMCs. Cracks were observed within IMC particles in the bulk solder, along the boardside and component side interfaces, and in the bulk solder traveling toward voids. Components with joint Au contents higher than 10 wt% had unacceptably poor reliability. The conclusion of this work is that gold content of SAC305 solder joints on boards with Au over Ni surface finishes should be kept below 3 wt% to conservatively minimize the risk of creating a microstructure that has poor reliability.

An isothermal calorimeter controlled by the Peltier effect has been designed and constructed in order to investigate reaction rates in solventwater mixtures. Because a thermal method was used a constant temperature environment was essential and this was achieved by using a water bath controlled to + 0.0010C. This callorinieter has been used to study the alkaline hydrolysis of methyl acetate in dimethylsulphoxide, and tetrahydrofuran - water mixtures at 15, 25 and 35 [degrees]C. The results of other investigations on similar reactions have been reviewed and an attempt has been made to correlate the electrostatic theories of Laidler and Eyring, and Amis and jaffe with these results. Finally, because it appears that specific solvent interactions play a major part in the reaction rates the role of water in the reaction mechanism has been examined. A mechanistic explanation has been proposed in order to correlate the rate of reaction with the composition of water-solvent mixtures which justifies the Laidler and Eyring treatment of solvent effects on ion-molecule reactions. / Bradford University