Thèses sur le sujet « MBW complex »

Submitted by EDUARDO RIBEIRO PINTO (eduribeiro2@bol.com.br) on 2018-05-03T15:47:28Z No. of bitstreams: 1 dissertacao_Eduardo.pdf: 6068904 bytes, checksum: 4ff00adcd4667c6d7ed4bcfb5db2321a (MD5)
Approved for entry into archive by Sulamita Selma C Colnago null (sulamita@btu.unesp.br) on 2018-05-03T19:01:49Z (GMT) No. of bitstreams: 1 pinto_er_me_bot_int.pdf: 6068904 bytes, checksum: 4ff00adcd4667c6d7ed4bcfb5db2321a (MD5)
Made available in DSpace on 2018-05-03T19:01:49Z (GMT). No. of bitstreams: 1 pinto_er_me_bot_int.pdf: 6068904 bytes, checksum: 4ff00adcd4667c6d7ed4bcfb5db2321a (MD5) Previous issue date: 2018-02-23
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Os Modelos Baseados em Indivíduos (MBI’s) têm sido crescentemente empregados na modelagem de processos infecciosos. Um MBI consiste de uma estrutura na qual ocorrem interações entre um certo número de indivíduos, cujo comportamento é determinado por um conjunto de características que evoluem estocasticamente no tempo. Estudos recentes têm mostrado que as redes complexas constituem um suporte natural para o estudo da propagação de uma doença. Redes complexas são descritas por um conjunto de vértices (nós), arestas (conexões, ligações ou links) e algum tipo de interação entre os mesmos. Na formulação original do MBI e em modelos SIR (Suscetível, Infectado e Recuperado) e SEI (Suscetível, Exposto e Infectado), as relações entre os indivíduos são representadas por grafos completos, ou seja, todos os indivíduos estão conectados entre si. Como a topologia de uma rede real não pode ser descrita por uma rede puramente aleatória, nesse trabalho o MBI foi implementado de forma a incorporar modelos mais realísticos de redes de contato na propagação de uma doença infecciosa. De maneira geral, observou-se que redes complexas com diferentes topologias resultam em curvas de indivíduos suscetíveis, infectados e recuperados (ou suscetíveis, expostos e infectados) com diferentes comportamentos, e desta forma, que a evolução de uma dada doença, em particular a tuberculose, é altamente sensível à topologia de rede utilizada. Mais especificamente, observou-se que quanto maior o valor do comprimento do salto médio, mais rápida será a propagação da doença e, consequentemente, maior será o número de indivíduos infectados.
Individual-Based Models have been increasingly employed in the modeling of an infectious process. An IBM consists of a structure in which interactions occur between a certain number of individuals, whose behavior is determined by a set of characteristics that evolve stochastically in time. Recent studies have shown that complex networks are a natural framework for the study of a disease spread. Complex networks are described by a set of vertices (or nodes), edges (connections or links) and some type of interactions between them. In the original IBM approach and in SIR (Susceptible, Infected, Recovered) and SEI (Susceptible, Exposed and Infected) models, the relations between individuals are represented by complete graphs, that is, all individuals are connected to each other. Since the topology of a real network can not be described by a purely random network, in this work an IBM has been implemented in order to incorporate some realistic contact networks xvii models. In general, it was observed that complex networks with different topologies correspond to curves of susceptible, infected and recovered individuals (or susceptible, exposed and infected) with different behaviors, and thus, that the evolution of a given disease, in particular tuberculosis, is highly sensitive to a network topology. More specifically, it was observed that the higher the value of the mean jump length is, the faster the disease spreads and consequently, the higher is the number of infected individuals.

In eukaryotic cells a key regulatory step of cell cycle occurs in late G1, which has been termed “Start” in yeast cells. At this point, cells determine whether they will go to through a new round of proliferation, or choose alternative pathways; cell cycle arrest or sexual differentiation. In fission yeast, the MBF complex controls the transcriptional activation of some genes of G1/S transcriptional wave, including cdc18, cdt1 (necessary to prevent onset of mitosis before of completion of DNA synthesis), and cdc22 (ribonucleotide reductase). Control of MBF activity is essential for normal cell cycle progression. It has been found that MBF complex is bound to the promoter of its target genes throughout the cell cycle, implicating that MBF activity is not regulated by pure binding to DNA. We purified novel inetractor of MBF complex, INO80 complex. Here we demonstrate that INO80 regulates cell cycle genes through MBF, and that proper acetylation of histone variant H2A.Z is crucial for MBF dependent transcription.
En las células eucariotas el paso clave en la regulación de ciclo celular ocurre en el final de la fase G1, nombrado como “Start” en levadura. En este punto, las células determinan si pasaran por nueva ronda de la proliferación, o elegirán vías alternativas: parada del ciclo celular o diferenciación sexual. En S. pombe, el complejo MBF controla la activación de la transcripción de algunos genes necesarios para la transición G1 / S, incluyendo cdc18, cdt1 (necesario para evitar el inicio de la mitosis antes de la finalización de la síntesis de ADN), y cdc22 (ribonucleótido reductasa). El control de la actividad MBF es esencial para la progresión normal del ciclo celular. El complejo MBF se une a los promotores de los genes, a lo largo del ciclo celular, implicando que la actividad de MBF no esta regulada por el simple hecho de unión al DNA. Hemos purificado un interactor nuevo de MBF, el complejo INO80. En esta tesis demostramos que INO80 regula los genes del ciclo a través de MBF, y que la acetilación adecuada de la histona H2A.Z es crucial para la transcripción de los genes MBF.

The nucleosome remodeling and deacetylase (NuRD) complex is an abundant deacetylase complex, which couples histone deacetylation and chromatin remodeling ATPase activities, and has a broad cellular and tissue distribution. Although the working model of how this complex forms and functions is not well known, we have demonstrated that the coiled-coil interaction between two proteins (MBD2 and p66α) is critical for DNA methylation dependent gene silencing in vivo. Chapter one: ‘Unique features of the anti-parallel, heterodimeric coiled-coil interaction between methyl-cytosine binding domain 2 (MBD2) homologues and p66α dictate high affinity binding’ describes this unique coiled coil interaction. Coiled-coils were studied using a variety of biophysical techniques including analytical ultracentrifugation (AUC), isothermal titration calorimetry (ITC) and circular dichroism (CD). Results were compared across homologues and mutation studies were carried out to test our hypotheses. The studies reported in this chapter add to our understanding of coiled-coil interaction and thereby facilitate development of small peptide based drugs which target such interactions in nature.A number of proteins have been identified in humans that specifically bind to methylated CpG via a methyl binding domain (MBD). The human genome encodes at least five MBD proteins: MeCP2 and MBD1 through MBD4, which are homologous in their methyl binding domains but not many similarities are seen outside the MBD. Out of the five MBDs, MBD4 has a c-terminal glycosylase domain through which it recognizes mCpG.TpG mismatch and is important for base excision repair system. Chapter two: ‘Dynamic behavior of MBD4 in methylated DNA recognition’ focuses on MBD4 and its preference for DNA methylation mark. Techniques of surface plasmon resonance (SPR), nuclear magnetic resonance (NMR) spectroscopy are used to study binding affinity for variations of methylated DNA mark. Chemical exchange studies are used to demonstrate how MBD4 scans for methylation mark and these studies have added a new dimension to our understanding of how MBD proteins ‘read’ DNA methylation marks. Chapter three: ‘Solving the solution structure of MBD domain of MBD4 on methylated DNA by NMR’ describes a process of structure determination using NMR spectroscopy. The focus of this chapter is not on developing a new technique but rather on using current resources to solve a protein structure, which can be used to further understand our biological system. Here, I have discussed the workflow used to determine a final three-dimensional structure starting from sample preparation, data collection, data analysis to structure calculation.

Le contrôle combinatoire de l’ expression des gènes est une caractéristique importante du profil spatio-temporel de l'accumulation des flavonoïdes chez les plantes. Des résultats précédents avaient démontré chez Arabidopsis thaliana, que la régulation de l’accumulation des anthocyanes et des proanthocyanidines repose sur l'activité de facteurs de régulation de la transcription de type R2R3-MYB et bHLH qui forment des complexes ternaires ("MBW") avec la protéine TTG1 (WDR). L'objectif de la thèse était de caractériser les complexes MBW impliqués et leurs cibles, pour avoir une compréhension globale des mécanismes transcriptionnels qui contrôlent la biosynthèse des flavonoïdes.En utilisant différentes approches génétiques et moléculaires, nous avons montré que seuls les gènes « tardifs » (c’est à dire DFR, LDOX, BAN, TT19, TT12 et AHA10) sont des cibles directes des complexes MBW. Bien que le complexe de régulation impliquant les protéines TT2-TT8-TTG1 ait un rôle majeur dans la régulation de ces gènes structuraux dans la graine d’Arabidopsis, trois autres complexes contenant MYB5, GL3 ou EGL3 sont également impliqués de façon tissu-spécifique. Comme l’expression du gène TT8 joue un rôle clef dans ces régulations, une dissection fonctionnelle de son promoteur a été entreprise. Elle a montré la nature modulaire de ce promoteur avec deux domaines impliqués dans l’expression tissu-spécifique et un troisième dans la force du promoteur. Les résultats obtenus suggèrent également l’existence d’autres régulateurs qui restent à caractériser. Enfin, nous avons développé une nouvelle technique de caractérisation des interactions entre les facteurs de transcription et les promoteurs, basée sur l’expression transitoire dans des protoplastes de mousse (i.e. Physcomitrella patens). Nous avons ainsi pu identifier les éléments cis des promoteurs impliqués dans la régulation de l’expression de TT8 et de chacun des promoteurs cibles des complexes MBW.L’ensemble de ces travaux permet de fournir un modèle plus complet du réseau de régulations transcriptionnelles qui contrôle la biosynthèse des proanthocyanidines et des anthocyanes, ainsi que des outils et de nouvelles pistes pour poursuivre ces études chez Arabidopsis et d’autres plantes
The combinatorial control of gene expression is a key feature of the spatio-temporal pattern of flavonoid accumulation in plants. Previous results have shown that the regulation of anthocyanins and proanthocyanidins (PAs or tannins) pigmentation relies on the transcriptional activity of R2R3-MYB and bHLH proteins that form “MBW” ternary complexes with TTG1 (WD-Repeats), in Arabidopsis thaliana. The purpose of the thesis was to figure out the nature and spatio-temporal activity of these MBW complexes and to identify their direct targets, which were essential steps toward a comprehensive understanding of the transcriptional mechanisms that control flavonoid biosynthesis. Using different molecular and genetic approaches this thesis has demonstrated that only late biosynthetic genes (namely DFR, LDOX, BAN, TT19, TT12 and AHA10) are direct targets of the MBW complexes. Interestingly, although the TT2-TT8-TTG1 complex was shown to play the major role in regulating the expression of these structural genes in developing seeds, three additional MBW complexes that contain MYB5, GL3 or EGL3 are also involved, in a tissue-specific manner. Because the expression of TT8 is largely involved in these regulations, a functional dissection of its promoter was carried out. Two modules drive the tissue-specific activity of the TT8 promoter in PA- and anthocyanin-accumulating cells, and a third module is responsible for the strength of the promoter. Interestingly, this regulation involves at least six different MBW complexes. Our results also suggest that some putative new regulators remain to be discovered. Last, use of a newly developed fast and sensitive transient expression system that relies on protoplasts of the moss Physcomitrella patens has allowed the identification of both, specific cis-regulatory elements through which TT8 expression is regulated and the minimal promoter for each of the genes that are targeted by the MBW complexes.Altogether, by answering fundamental questions and by demonstrating or invalidating previously made hypotheses, we have provided a new and comprehensive view of the regulatory mechanisms controlling PA and anthocyanin biosynthesis in Arabidopsis, as well as new clues and tools for further investigation of this pathway in Arabidopsis and other plant species

La localització de molècules d'ARNm permet regular la síntesi de proteïnes localment i temporalment i això és vital per molts processos cel·lular fonamentals. A Oligodendròcits el transport de l'ARNm de la proteïna bàsica de la mielina (MBP) permet la síntesi local d'aquesta. MBP té una funció essencial en la formació de la beina de mielina que envolta els exons, nodreix les cel·les nervioses i permet la rapida transmissió dels estímuls. Tot i la intensa recerca feta sobre els mecanismes moleculars que condueixen el transport i l'expressió local MBP-ARNm, encara no està clar com l'ARNm és transportat fins al seu destí i com es controla la traducció de la proteïna al lloc adequat. Fent servir informació de dades publicades i el cribratge d'interaccions entre proteïnes realitzat al laboratori hem seleccionat dos complexos proteics essencials per a la localització de l'ARNm de la MBP i hem intentat construir-los des de la base. Des d'aquest enfocament la reconstrucció in vitro dels complexos ens permet analitzar la funcio de cada component del complex i entendre més profundament com es controla aquest procés biològic. La síntesi de l'ARNm de la MBP és parcialment depenent de la interacció entre la proteïna d'unió a ARN, hnRNPA2, i una polimerasa de microtúbuls, chTOG. Per aclarir el mecanisme pel qual aquesta interacció desencadena la traducció de la MBP vam decidir analitzar les conseqüències funcionals d'aquesta nova interacció entre una proteïna d'unió a microtúbul i una proteïna d'unió a ARN. He aconseguit amb èxit proteïna recombinant funcional de hnRNPA2 I chTOG. La interacció entre aquestes dues proteïnes va resultar no especifica. Hipotetitzo que és degut a la conformació que adquireix la proteïna hnRNPA2 en els grànuls de ribonucleoproteines o depenent d'altres components. Degut a la falta de recursos per investigar-ho vam suspendre aquest projecte. El procés de lliurament de l'ARNm de MBP a oligodendròcits és mantingut per kinesines dependents de microtúbuls. La kinesina Kif1Bβ s'ha vist que fa el transport però els adaptadors que uneixen l'ARNm al motor(kinesina) resten desconeguts. Kif1Bβ se sap que és responsable del transport d'orgànuls de membrana com precursors de vesícules sinàptiques. Evidencies de la bibliografia i del cribratge d'interaccions entre proteïnes realitzat al laboratori suggereixen que Kif1Bβ podria realitzar el transport de l'ARN per captura de les vesícules. La regulació de les kinesines no és del tot conegut per això vaig decidir investigar primer aquest aspecte. Per aquesta raó vaig provar de construir i caracteritzar el sistema de transport de vesícules depenent de Kif1Bβ in vitro. Vaig aconseguir produir i caracteritzar la proteïna recombinant Kif1Bβ. Kif1Bβ es capaç de formar un dímer en solució de forma independent i en aquest estat de dímer moure's al llarg de microtúbuls. Vaig detectar la preferència de Kif1Bβ per certs lípids que després vaig fer servir per produir vesícules sintètiques. Les vesícules es van fer servir per caracteritzar in vitro el seu transport per Kif1Bβ. La reconstrucció de les interaccions entre proteïnes implicades en el transport de complexos van demostrar ser més problemàtiques. Esforços addicionals serien necessaris en el futur per aconseguir aquest repte i millor caracterització del proposat complex. Tot i els meus esforços no vaig poder reconstruir completament cap dels dos complexos d'interès. Tot i així, presento en aquesta tesi una descripció detallada dels enfocaments i els complexes parcials que nosaltres vam aconseguir construir i caracteritzar.
Localization of mRNA molecules enables locally and temporally regulated protein synthesis, which is vital for many fundamental cellular processes. In oligodendrocytes transport of myelin basic protein (MBP) mRNA to myelinating processes allows for local synthesis of MBP. MBP has an essential function if forming the myelin sheath that surrounds axons, nurtures the nerve cells and allows for fast stimuli transmission. Despite extensive research about the molecular mechanisms that guide the transport and local expression of MBP-mRNA it is still not clear how this mRNA is transported to its destination and how activation of translation at the right place is controlled. Using the information from published data and an interaction screen performed in the lab we selected two protein complexes essential for MBP mRNA localisation and attempted to build them from bottom-up. In this approach in vitro reconstitution of the minimal complexes allows us to analyse the function of each component of the complex and understanding better how biological processes are regulated. MBP mRNA synthesis is partially dependent on the interaction between an RNA binding protein, hnRNPA2, and a microtubule polymerase, chTOG. To elucidate the mechanism by which this interaction triggers MBP mRNA translation we decided to analyse the functional consequences of this entirely novel interaction between a microtubule binding and RNA binding protein. I successfully produced functional recombinant hnRNPA2 and chTOG. The interaction detected between those two proteins turned out to be unspecific. I hypothesize it is due to the conformation that hnRNPA2 acquires in the RNP granule or depends on other components. Due to the lack of resources to investigate this further we suspended this project. Delivery of MBP mRNA to the oligodendrocytes processes is maintained by microtubule-dependent kinesin motors. The kinesin Kif1Bβ has been shown to carry out the transport but the adaptors, which link the mRNA to the motor, remain unknown. Kif1Bβ is known to be responsible for transport of membranous organelles such as synaptic vesicle (SV) precursors. Evidence from literature and protein interaction screen performed in the lab suggests that Kif1Bβ could achieve mRNA transport by vesicle hitchhiking. The regulation of the kinesin is not fully understood therefore I decided to investigate this aspect first. For this reason I sought to build and characterize a Kif1Bβ-dependent vesicle transport system in vitro. I produced recombinant protein and achieved characterization of the Kif1Bβ motor. Kif1Bβ is able to independently form a dimer in solution and in the dimeric state processively moves on microtubules. I detected the preference of Kif1Bβ for certain lipids, which were then used to produce synthetic vesicles. In in vitro motility assays I characterized the Kif1Bβ-driven transport of synthetic vesicles. The reconstitution of the interactions between proteins involved in vesicle transporting complex proved to be more problematic. Additional efforts are needed to achieve this goal and further characterize the proposed complex in the future. Despite my efforts I did not manage to fully reconstitute any of the two complexes of interest. However, I report in this thesis a detailed description of the approaches and the partial complexes I managed to build and characterise.

Corporations seek Master of Business Administration (MBA) students who are ready to perform upon hiring. Business schools need to align instructional practices and technology with student, accreditation, and marketplace demands. Complex simulation use has increased exponentially to provide MBA students with business experience in the classroom. Methods to assess the effectiveness of complex simulations to achieve learning outcomes is limited to student perceptions of learning, satisfaction, and direct assessment separately. The purpose of this quantitative ex post facto comparative study was to examine MBA students’ perception of learning to real performance in integrative courses with complex simulation. Archival MBA student Peregrine COMP™ pretest, posttest, and SIRII™ scores were analyzed using independent t-test, paired sample t-test, and Pearson r coefficient. MBA students perceived higher levels of learning in courses with complex simulation based on the statistically significant increase in SIRII™ scores over courses without simulation. Another key finding from the quantitative study was the statistically significant negative correlation of students’ perception of learning to actual performance. Positive student perceptions of learning could hide a complex simulation’s inability to meet student learning outcomes, according to the statistically significant decrease between pretest, and posttest Peregrine COMP™ scores. Based on the quantitative correlation analysis of student perceptions of learning to actual performance, MBA administrators and faculty need to evaluate the use of instructional technology from multiple data points to avoid applications that offer minimal value to achieving learning outcomes. Future research opportunities could include a larger MBA population from multiple regions of the United States. Additional studies could investigate undergraduate perceptions of learning to actual performance to assess any benefit from complex simulations.

Research interest in complex oxides has resurged owing to progress in modern epitaxial techniques. Among such oxides, lead-titanate-based thin films such as PbTiO3 (PTO) and Pb(ZrxTi1−x)O3 (PZT) offer attractive advantages for a wide variety of applications. Moreover, integration between functional oxides with compound semiconductors has the potential to realize multi-functional devices which enjoy the properties from both groups of materials. Ferroelectric materials with a perovskite structure (ABO3) and semiconductors such as GaN with a hexagonal structure, require a careful choice of a bridge layer and suitable epitaxial technique. Molecular beam epitaxy (MBE) has been an established technique in providing epitaxial growth with high crystal perfection and precise control over material composition. Single-crystal oxides grown by molecular beam epitaxy (MBE) can in principle avoid grain boundaries and provide a sharp interface as well. In this dissertation, the MBE growth mechanism of PZT was investigated. In-situ RHEED patterns indicate that the growth of PTO and PZT occur in a two-dimensional, layer by layer mode, as confirmed by a streaky pattern. The crystal quality of PTO, PZO, and PZT thin films prepared by MBE are evaluated by X-ray diffraction (XRD), and have a full width at half maximum (FWHM) value of 4 arcmin for an 80nm thick layer. Optical properties of the PTO thin films have been characterized by variable angle spectroscopic ellipsometry (VASE), and well resolved dielectric functions are extracted. The refractive index is determined as 2.605 at 633 nm, and bandgap energy as 3.778eV. The electrical properties of the PTO and PZT are evaluated by the measurement of polarization-field hysteresis loops, give a remanent polarization of 83 μC/cm2 and a coercive field of 77 kV/cm. Lead oxide (PbO), titanium dioxide (TiO2), and zirconium dioxide (ZrO2), on GaN templates for potential PZT/GaN integration. The epitaxial growth of TiO2, PbO, and ZrO2 is realized on GaN templates for the first time by MBE. The PbO epitaxial layer was also used as a nucleation layer to enable single crystalline, perovskite PTO growth on GaN.

Oxide related research has increased as standard oxides reach their operational limits and new classes of devices are imagined that can only be realized through the use of man-made compounds. Many of these devices require high quality films in order to reach their highest potential. Molecular beam epitaxy (MBE) is poised to become a key producer of high quality oxides. One of the most promising oxides is lithium niobate, LiNbO3, which can potentially deliver novel electronic, optic, and hybrid devices not currently possible. Growing lithium niobate using MBE is difficult. Several concepts are presented that will make this task easier. First, high temperature refractory metals can be delivered to the substrate through a novel use of low temperature chloride compounds such as niobium (V) chloride. This chloride chemistry allows low temperature sources to deliver high temperature materials to the substrate. Second, a precision, vapor-phase source and control system is prototyped for these chloride compounds achieving improved flux accuracy and expanding the capability of standard MBEs to support many sources. Chloride sources have high vapor pressures and are sensitive to temperature changes causing flux drift. The vapor-phase source removes the temperature sensitivity and eliminates thermal drifts. Third, a novel method of measuring flux with spontaneous ionzation current has been developed. This design utilizes a low noise design to measure femtoamp currents generated as an evaporant spontaneously ionizes. The measured current with additional predicted data has the potential for directly counting the atoms evaporated and controlling evaporation from a source. The design is sensitive enough to detect outgassing of the cell and cell "spitting" or other non-idealities. Monitoring these non-idealities can help improve other processes by ensuring the cell is fully outgassed and stable. Finally, a miniaturized RF induction cell prototype is shown that can eliminate the need for incandescent filaments in an oxide based MBE. The RF cell has the potential to increase reliability of MBEs for oxide work and achieve higher operating temperatures without the need for densely wound incandescent filaments or electron beam sources.

Cette thèse concerne le développement d’approches spectroscopiques infrarouge et Raman exaltées de surface: la spectroscopie infrarouge exaltée de surface (SEIRAS) combinée avec une cellule de perfusion et la spectroscopie Raman exaltée de surface (SERS) couplée avec l’électrochimie. Dans le cadre du premier projet, différentes protéines ont été étudiées : lactose perméase (LacY), complexe I et IM30. Nous avons déterminé le pKa de Glu325 dans LacY sauvage et dans différents mutants portant des mutations dans le centre actif de translocation des protons. Sauvage complexe I a été oxydé avec différents agents oxydants et réduit avec NADH. Spectres différentiels correspondants ont été analysés. Des changements conformationnels dans la protéine IM30, induits par la présence des ions Mg2+, ont été observés.Dans le cadre du deuxième projet, une cellule spectroélectrochimique contenant une grille d’or a été adaptée pour étudier des protéines redox actives. Cette grille d’or sert à la fois de substrat SERS et d’électrode de travail. Cyt c, Hb et Mb ont d'abord été utilisés pour valider la configuration, puis l'approche a été étendue pour étudier une protéine membranaire
This thesis concerns the development of surface-enhanced infrared and Raman spectroscopic approaches: surface-enhanced infrared absorption spectroscopy (SEIRAS) combined with perfusion cell and surface-enhanced Raman spectroscopy (SERS) combined with electrochemistry. Within the first project different proteins were studied: Lactose Permease (LacY), complex I and IM30.The pKa of Glu325 in LacY WT and in different mutants carrying mutations in the proton translocation active center was determined. WT complex I was oxidized with different oxidizing agents and reduced with NADH. Corresponding redox-induced conformational changes were studied. The evidence was given that Mg2+ ions induce conformational changes in the protein IM30.Within the second project the spectroelectrochemical cell containing gold grid electrode was adopted for the studies of redox active proteins. This gold grid serves both as working electrode and as SERS active substrate. First Cyt c, Hb and Mb were used to validate the setup and then the approach was extended to study a membrane protein

The thesis consists of two parts. Part 1 deals with the preparation of thin films and sub-micron powders of complex metal oxides by nebulized spray pyrolysis (NSP) and Part 2 consists of Brillouin scattering studies of solid materials exhibiting interesting phase transitions. The simple technique of NSP has been employed to prepare thin films of A12O3, PbTiO3, Pb(Zr0.5Ti0.5)O3 (PZT) and PbZrO3 on single crystal substrate. The films were characterized by various techniques for their composition, structure, morphology and dielectric properties. Ferroelectric (FE) films of the configuration FE/LaNiO3/SiO2/Si (FE = PbTiO3 and PZT), wherein the LaNiO3 barrier electrode was also deposited on the SiO2/Si substrate by NSP, have been investigated. The films exhibit satisfactory ferroelectric properties. PbZrO3 films deposited on LaNiO3/SiO2/Si substrates show good features, including a reversible AFE ↔ FE transition. Sub-micron particles of TiO2, ZrO2, Pb(Zr0.5Ti0.5)O3, Al2O3, S1O2 and mullite have been prepared by NSP and characterized by various techniques. Brillouin scattering has been used, for the first time, not only to characterize the Peierls transition but also the incommensurate to commensurate transition in the one-dimensional blue bronze, K0.3M0O3. The charge density wave transition in NbSe2 has also been investigated by Brillouin scattering. The charge ordering and antiferromag-netic transitions in single crystals of the rare earth manganates, Nd0.5Ca0.5MnO3 and Pr0.63Ca 0.37MnO3, have been investigated by Brillouin scattering. It is noteworthy that the temperature variation of the Brillouin shift and intensity parallel to that of the magnetization, thereby throwing light on magnetic excitations in charge-ordered state. Brillouin scattering investigations of C60 and C70 films have yielded values of the elastic moduli.

The complement system is part of the innate immune system and is crucial for identifying invading microorganisms. The lectin pathway of complement activation is initiated through multimeric macromolecules which recognise pathogen-associated patterns and translate binding through activation of associated serine proteases that start a cascade of proteolytic events leading to bactericidal, opsonising and proinflammatory responses. Mannan-binding lectin (MBL) is one of the macromolecules mediating binding to specific carbohydrate structures common to a range of microorganisms. Three different mannan-binding lectin associated serine proteases (MASP-1/-2/-3) and a non-enzymatic protein of 19 kDa (MApl9) have been described. MASP-2 appears to mediate all processes required for complement activation while little or no complement-related functional activity was found to be mediated by MASP-1, MASP-3 or MApl9. Functional and biophysical studies of MASP-3 relied on continuous and reliable supply of recombinant MASP-3. In this work production of recombinant MASP-3 in mammalian cells with subsequent affinity purification and characterisation of the recombinant MASP-3 was performed to obtain large quantities of homogeneous enzyme. The development of screening assays and assays for quantitative determination of MASP-3 levels were two other tools essential for the development of this thesis. As a result of this thesis MASP-3 levels in different body fluids were determined in healthy individuals using a quantitative assay. The assays were used to analyse the MASP-3 level in sample collections from patients suffering from Alzheimer disease and type-1 diabetes. The correlation of MASP-3 level and MBL genotype, H-/L-Ficolin concentration, age, BMI, acute phase and time of year were analysed. The enzymatic activity of MASP-3 was analysed on chromogenic substrates and the results permitted a study of MASP-3 inhibition. Attempts were made to affinity purify potential MASP-3 substrates and ligands using beads coupled with recombinant MASP-3 and anti- MASP-3 antibodies. The influence of calcium on MASP-3 complex formation, dissociation, activation and stability was analysed.

The multiple constant multiplication (MCM) operation is a fundamental operation in digital signal processing (DSP) and digital image processing (DIP). Examples of the MCM are in finite impulse response (FIR) and infinite impulse response (IIR) filters, matrix multiplication, and transforms. The aim of this work is minimizing the complexity of the MCM operation using common subexpression elimination (CSE) technique and redundant number representations. The CSE technique searches and eliminates common digit patterns (subexpressions) among MCM coefficients. More common subexpressions can be found by representing the MCM coefficients using redundant number representations. A CSE algorithm is proposed that works on a type of redundant numbers called the zero-dominant set (ZDS). The ZDS is an extension over the representations of minimum number of non-zero digits called minimum Hamming weight (MHW). Using the ZDS improves CSE algorithms' performance as compared with using the MHW representations. The disadvantage of using the ZDS is it increases the possibility of overlapping patterns (digit collisions). In this case, one or more digits are shared between a number of patterns. Eliminating a pattern results in losing other patterns because of eliminating the common digits. A pattern preservation algorithm (PPA) is developed to resolve the overlapping patterns in the representations. A tree and graph encoders are proposed to generate a larger space of number representations. The algorithms generate redundant representations of a value for a given digit set, radix, and wordlength. The tree encoder is modified to search for common subexpressions simultaneously with generating of the representation tree. A complexity measure is proposed to compare between the subexpressions at each node. The algorithm terminates generating the rest of the representation tree when it finds subexpressions with maximum sharing. This reduces the search space while minimizes the hardware complexity. A combinatoric model of the MCM problem is proposed in this work. The model is obtained by enumerating all the possible solutions of the MCM that resemble a graph called the demand graph. Arc routing on this graph gives the solutions of the MCM problem. A similar arc routing is found in the capacitated arc routing such as the winter salting problem. Ant colony optimization (ACO) meta-heuristics is proposed to traverse the demand graph. The ACO is simulated on a PC using Python programming language. This is to verify the model correctness and the work of the ACO. A parallel simulation of the ACO is carried out on a multi-core super computer using C++ boost graph library.

碩士
國立中興大學
生物化學研究所
96
T2SS of Xanthomonas campestris pv campestris is assembled by 12 proteins. XpsE is the only cytoplasmic component and the likely energy supplier of the system, whereas XpsL is a bitopic membrane protein with a single transmembrane segment. The role of XpsL in T2SS is not so clear. It has been previously observed that the hexameric XpsE, whose formation is nucleotide-dependent, interacts in vitro directly with the cytoplasmic domain of XpsL as MBP-XpsLN. We thus speculated that XpsE may form complex with XpsLN in vivo. In this study, we attempted the complex isolation by coexpressing XpsE and MBP- XpsLN in E. coli. Copurification of MBP-XpsLN and Strep-tagged XpsE was observed on the SDS-PAGE when purified using double-affinity chromatography, indicating that a stable XpsE/MBP-XpsLN complex was formed as a consequence of their coexpression in E. coli. The molecular size of such a complex was estimated to be 800 kDa as revealed by size-exclusion chromatography. We thus postulated that the complex may be constituted of 6 molecules each component. The protein complex purified from size-exclusion chromatography exhibited an ATPase activity sixfold that of the singly expressed XpsE. In addition, the ATPase activity of the complex was stimulated by cardiolipin by threefold. The XpsE/MBP-XpsLN complex resulted from the coexpression strategy employed here might resemble an intermediate stage during secretion process in vivo, thus enabling us to study in the future the mechanistic events driven by the interaction between XpsE and XpsL.

A major challenge faced by project managers is balancing the variables of scope, cost, and schedule. Changes in scope usually result in cost/schedule overruns. Variance in either or both of them creates disorder (typically increases) in the estimated or projected time and cost. Therefore, controlling cost and schedule are two of the most critical aspects of a construction project. This research uses two already existing management theories, specifically Management by Means (MBM) and Management by Results (MBR), and analyzes a case where these two theories are combined with the goal of improving construction practices. This research compares an eight month schedule in a construction project and relates Percentage of Planned activities Completed (PPC) with projected and actual draw (cash) calls. The research analyzes the question of how lean construction PPC captures variance in cost. The research method is based on a literature review, data collection, case study and data interpretation to answer the hypothesis that improvement in PPC over a particular month has a positive correlation with difference between cash calls. Because this research is limited to a time frame of 8 months in a single project, it is not statistically significant. However, this research serves to create a model template or pilot study for a larger study.

The presented thesis includes investigations to fully characterize the electronic structure and magnetic properties ofselected manganese containing high-spin molecules by means of various X-ray spectroscopic, magnetic and theoretical methods. The investigations on the Mn4 star-shaped molecule havelead to a number of interesting results. Magneto-chemical studies exhibit very weak exchange coupling constantsbetween the four Mn(II) ions, leading to complicated low lying states in which the ground state is not well separated, resulting from a dominant weak ferromagnetic coupling and a giant moment of up to 20 µB/f.u. XMCD measurements revealed that almost the completemagnetic moment is located around the Mn(II) ions.This is in agreement with only a few charge transfer states foundwithin the detailed X-ray absorption spectroscopic study. The electronic structure and detailed magnetic properties of the star-shaped heteronuclear CrIIIMnII3 complex have been precisely investigated.With XPS the homovalency of Mn and Cr have been verified. The XA-spectra of the manganese and chromium L edges were measured and compared to earlier investigated Mn4 spectra.The combination high-magnetic field magnetic measurements and element selective XMCD of Mn and Cr L edges and quantum model calculations lead to a complete analysis of the magnetic structure of the CrMn3 magnetic core. The III valence state of the manganese ions in MnIII6O2Salox has been verified. From X-ray diffraction, typical Jahn-Teller distorted oxygen octahedra have been found for Mn(III) ions. Comparisons of XPS and XAS spectra of the complex to corresponding spectraof maganite and tetranuclear manganese(II) cluster it was definitely possible to identify MnIII6O2Salox as a pure Mn(III) compound.

Packaging is an important element responsible for brand growth and one of the main rea-sons for producers to gain competitive advantages through technological innovation. In this re-gard, the aim of this work is to design a fully autonomous electronic system for a smart bottle packaging, being integrated in a European project named ROLL-OUT. The desired application for the smart bottle is to act as a fill-level sensor system in order to determine the liquid content level that exists inside an opaque bottle, so the consumer can exactly know the remaining quantity of the product inside. An in-house amorphous indium–gallium–zinc oxide thin-film transistor (a-IGZO TFT) model, previously developed, was used for circuit designing purposes. This model was based in an artificial neural network (ANN) equivalent circuit approach. Taking into account that only n-type oxide TFTs were used, plenty of electronic building-blocks have been designed: clock generator, non-overlapping phase generator, a capacitance-to-voltage converter and a comparator. As it was demonstrated by electrical simulations, it has been achieved good functionality for each block, having a final system with a power dissipation of 2.3 mW (VDD=10 V) not considering the clock generator. Four printed circuit boards (PCBs) have been also designed in order to help in the testing phase. Mask layouts were already designed and are currently in fabrication, foreseeing a suc-cessful circuit fabrication, and a major step towards the design and integration of complex trans-ducer systems using oxide TFTs technology.