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.

Tesis por compendio / [EN] The determination of genetic biomarkers is progressively becoming more extended and popular, being commercialized even in kits for personalized medicine. Establishing specific genotype variations for each patient, such as single nucleotide polymorphisms (SNPs), could be a fundamental tool in the field of diagnosis, prognosis and therapy selection. However, the use of DNA testing is not fully implemented in general healthcare, mainly due to technical and economic barriers associated to the current technologies, which are limited only to specialized centers and large hospitals. In this thesis, the main goal was to overcome these obstacles by developing simpler, faster and more affordable point-of-care (POC) genotyping systems. Allele discrimination was achieved by employing isothermal enzymatic reactions, like recombinase polymerase amplification (RPA), ligation of oligonucleotides and loop-mediated isothermal amplification (LAMP). These processes were integrated to colorimetric indicators and immunoenzymatic assays, in a microarray format. Using compact discs and polycarbonate chips as platforms, the detection was achieved through widespread electronics, like disc-reader, flatbed scanner and smartphone. To demonstrate their capacities, the resulting systems were applied for identifying SNPs in human samples, associated to therapies for tobacco smoking cessation, major depression disorder and blood clotting-related diseases. After selecting the proper conditions, all studied strategies discriminated SNPs in samples containing as low as 100 copies of genomic DNA, with an error rate below 15%. Most importantly, the developed methods have reduced assays times varying between 70 and 140 minutes, at a cost similar to a conventional PCR-based analog, but maintaining or raising amplification efficiency and eliminating the need of specialized temperature cyclers and fluorescence scanners. In conclusion, the biosensors based in isothermal reactions and consumer electronics devices greatly improve the competitivity of POC DNA analysis. It was demonstrated that the technologies developed in this thesis could support genotyping assays in low-resource areas, such as primary healthcare centers and emerging countries. Through this democratization of genetic testing and by performing adequate association studies, molecular diagnostics and personalized medicine practices could have their application extended to the clinical routine. / [ES] La determinación de biomarcadores genéticos es cada vez más extensa y popular, estando incluso comercializándose kits para medicina personalizada. Establecer las variaciones específicas en el genotipo de cada paciente, como los polimorfismos de un solo nucleótido (SNP) podría ser una herramienta fundamental en el campo del diagnóstico, pronóstico y selección de la terapia. Sin embargo, el uso de pruebas de ADN no se encuentra completamente implementado en la atención médica general, principalmente debido a las barreras técnicas y económicas asociadas a las tecnologías actuales, limitadas solamente a centros especializados y grandes hospitales. En esta tesis, el objetivo principal fue superar estos obstáculos mediante el desarrollo de sistemas de genotipado point-of-care (POC), más simples, rápidos y asequibles. La discriminación alélica se logró mediante el uso de reacciones enzimáticas isotermas, como la amplificación de la recombinasa polimerasa (RPA), la ligación de oligonucleótidos y la amplificación isotérmica mediada por bucle (LAMP). Estos procesos se integraron a indicadores colorimétricos y ensayos inmunoenzimáticos en formato de micromatriz. Utilizando discos compactos y chips de policarbonato como plataforma de ensayo, se ha logrado la detección mediante dispositivos electrónicos de consumo, como un lector de discos, escáner documental y teléfono móvil. Para demostrar sus capacidades, los sistemas resultantes se aplicaron a la identificación de SNPs en muestras humanas, asociados a terapias antitabaquismo, para depresión y enfermedades relacionadas con la coagulación de la sangre. Tras seleccionar las condiciones adecuadas, todas las estrategias estudiadas discriminaron SNPs en muestras conteniendo tan solo 100 copias de ADN genómico, con una tasa de error inferior al 15%. Más importante, los métodos desarrollados han reducido los tiempos de ensayo a valores entre 70 y 140 minutos, a un coste similar a un análogo convencional basado en la reacción en cadena de la polimerasa (PCR), pero manteniendo o aumentando la eficiencia de amplificación y eliminando la necesidad de termocicladores y escáneres de fluorescencia. En conclusión, los biosensores basados en reacciones isotérmicas y dispositivos de electrónica de consumo mejoran en gran medida la competitividad del análisis POC de ADN. Se ha demostrado que las tecnologías desarrolladas en esta tesis podrían apoyar los ensayos de genotipado en áreas de recursos escasos, como centros de atención primaria y países emergentes. A través de esta democratización de las pruebas genéticas y realización estudios de asociación adecuados, el diagnóstico molecular y las prácticas en medicina personalizada podrían extender su aplicación a la rutina clínica. / [CA] La determinació de biomarcadors genètics és cada vegada més extensa i popular, estant fins i tot comercialitzant-se kits per a medicina personalitzada. Establir les variacions específiques en el genotip de cada pacient, com els polimorfismes d'un sol nucleòtid (SNP) podria ser una eina fonamental en el camp del diagnòstic, pronòstic i selecció de la teràpia. No obstant això, l'ús de proves d'ADN no es troba completament implementat en l'atenció mèdica general, principalment a causa de les barreres tècniques i econòmiques associades a les tecnologies actuals, limitades solament a centres especialitzats i grans hospitals. En aquesta tesi, l'objectiu principal va ser superar aquests obstacles mitjançant el desenvolupament de sistemes de genotipat point-of-care (POC), més simples, ràpids i assequibles. La discriminació al·lèlica es va aconseguir mitjançant l'ús de reaccions enzimàtiques isotermes, com l'amplificació de la recombinasa polimerasa (RPA), la lligació de oligonucleòtids i l'amplificació isotèrmica mediada per bucle (LAMP). Aquests processos es van integrar a indicadors colorimètrics i assajos inmunoenzimàtics en format de micromatriu. Utilitzant discos compactes i xips de policarbonat com a plataforma d'assaig, s'ha conseguit la detecció mitjançant dispositius electrònics de consum, com un lector de discos, escàner documental i telèfon mòbil. Per a demostrar les seues capacitats, els sistemes resultants es van aplicar a la identificació de polimorfismes en mostres humanes, associats a teràpies antitabaquisme, per a depressió i malalties relacionades amb la coagulació de la sang. Després de seleccionar les condicions adequades, totes les estratègies estudiades van ser capaces de discriminar SNPs en mostres contenint tan sols 100 còpies d'ADN genòmic, amb una taxa d'error inferior al 15%. Més important, els mètodes desenvolupats han reduït els temps d'assaig a valors entre 70 i 140 minuts, a un cost similar a un anàleg convencional basat en la reacció en cadena de la polimerasa (PCR), però mantenint o augmentant l'eficiència d'amplificació i eliminant la necessitat de termocicladors i escàners de fluorescència. En conclusió, els biosensors basats en reaccions isotèrmiques i dispositius d'electrònica de consum milloren en gran manera la competitivitat de l'anàlisi POC del ADN. S'ha demostrat que les tecnologies desenvolupades en aquesta tesi podrien donar suport als assajos de genotipat en àrees de recursos escassos, com a centres d'atenció primària i països emergents. A través d'aquesta democratització de les proves genètiques i realització estudis d'associació adequats, el diagnòstic molecular i les pràctiques en medicina personalitzada podrien estendre la seua aplicació a la rutina clínica. / [PT] A determinação de biomarcadores genéticos está tornando-se cada vez mais extensa e popular, sendo comercializada até em kits para medicina personalizada. O estabelecimento de variações específicas de genotipo para cada paciente, tais como os polimorfismo de nucleotídeo único, pode ser uma ferramenta fundamental no campo do diagnóstico, prognóstico e seleção de terapias. No entanto, o uso de testes de DNA ainda não encontra-se totalmente implementado na área de saúde geral, principalmente devido às barreiras técnicas e econômicas associadas às tecnologias atuais, limitadas apenas a centros especializados e grandes hospitais. Nesta tese, o principal objetivo foi superar esses obstáculos desenvolvendo sistemas de genotipagem point-of-care (POC) de DNA, mais simples, rápidos e acessíveis. A discriminação de alelos foi alcançada empregando reações enzimáticas isotérmicas, como amplificação por recombinase polimerase (RPA), ligação de oligonucleotídeos e amplificação isotérmica mediada por loop (LAMP). Tais processos foram integrados a indicadores colorimétricos e ensaios imunoenzimáticos, em formato micromatriz. Usando discos compactos e chips de policarbonato como plataforma de ensaio, os analitos foram detectados através de dispositivos eletrônicos de consumo, como leitor de disco, scanner de mesa e smartphone. Para demonstrar suas capacidades, os sistemas resultantes foram aplicados para identificação de polimorfismos em amostras de DNA humano, associados a terapias antitabagismo, para depressão e doenças relacionadas à coagulação do sangue. Após a seleção das condições adequadas, todas as estratégias estudadas foram capazes de discriminar SNPs em amostras contendo até 100 cópias de DNA genômico, com uma taxa de erro inferior a 15%. Mais importante, os métodos desenvolvidos reduziram o tempo de ensaio a valores entre 70 e 140 minutos, com um custo similar a um método análogo baseado em reação em cadeia da polimerase (PCR), mas mantendo ou aumentando a eficiência da amplificação e eliminando a necessidade de cicladores de temperatura e scanners de fluorescência especializados. Em conclusão, os biosensores baseados em reações enzimáticas isotérmicas e dispositivos eletrônicos de consumo incrementam grandemente a competitividade da análise POC de DNA. Foi demonstrado que as tecnologias desenvolvidas nesta tese poderiam dar suporte a ensaios de genotipagem em lugares com poucos recursos, como centros de atenção primária e países emergentes. Através desta democratização dos testes genéticos e com a realização de estudos de associação adequados, o diagnóstico molecular e as práticas de medicina personalizada poderiam ter sua aplicação extendida à rotina clínica. / The authors acknowledge the financial support received from the Generalitat Valenciana (GVA-PROMETEOII/2014/040 Project and GRISOLIA/2014/024 PhD grant) and the Spanish Ministry of Economy and Competitiveness (MINECO CTQ2013-45875-R project) / Yamanaka, ES. (2020). Isothermal-based DNA biosensors for application in pharmacogenetics [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/148366 / TESIS / Compendio

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.