京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24947号 / 生博第509号 / 新制||生||68(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 原田 浩, 教授 松本 智裕, 教授 鈴木 淳 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

A presente pesquisa teve como objetivo estudar a resistência ao cisalhamento no plano de cola de corpos de prova especiais e avaliar o seu impacto na geometria do dente, na resistência à tração de emendas dentadas e nas propriedades oriundas da flexão de vigas simples e compostas. A resistência ao cisalhamento no plano de cola foi estudada segundo o seu ângulo de inclinação em relação às fibras, desde 0 até 90o na madeira de Eucalyptus grandis e Pinus elliottii. O Eucalyptus grandis foi favorecido por pequenos ângulos de inclinação do plano de cola contrariamente ao que aconteceu com o Pinus elliottii e, portanto, apresentou maior eficiência na colagem, o que é desejável para viga laminada colada. Os valores obtidos foram divididos pela resistência ao cisalhamento da madeira maciça para encontrarem-se as eficiências das ligações coladas com vários adesivos. O valor da resistência ao cisalhamento no ângulo de 5° foi utilizado conjuntamente com a resistência à tração da madeira maciça de Eucalyptus grandis para calcular o comprimento do dente a partir de uma teoria simples que o relaciona aos demais parâmetros já conhecidos ou simulados da emenda dentada. Essa teoria, resumida numa única identidade, foi desenvolvida para otimizar a emenda em termos de seus esforços solicitantes majoritários.A emenda dentada foi testada à tração paralela em lâminas emendadas com dentes verticais e horizontais. O perfil vertical apresentou maior resistência à tração e a diferença foi explicada pela área de colagem individual do dente. No caso do dente vertical essa área era menor e, portanto, penalizava menos quando havia algum defeito no dente. Na análise dos tipos de ruptura apresentados verificou-se que a ruptura do tipo 1 que denota má colagem por causa intrínseca do adesivo ou falta de pressão na colagem e ruptura do tipo 5 ou 6 que evidenciam pontos excessivamente fracos da barra ligada ou pontos de concentração de tensões imposta pelas garras da máquina no ensaio de tração contribuíam significativamente para redução da resistência à tração da emenda dentada. Essa resistência deve ser caracterizada por ocorrência alternada de rupturas por cisalhamento misto na superfície do dente e ruptura por tração na seção reduzida da barra ou na raiz do dente como acontece na ruptura do tipo 3. Na flexão foram ensaiadas lâminas emendadas trabalhando como viga sujeita a tensões normais de tração e de compressão e lâminas emendadas trabalhando como componente de viga laminada posicionada na borda tracionada. Não houve diferença entre esses dois casos em qualquer dos estados limites. A eficiência da viga laminada colada com emenda no meio do vão foi muito baixa em relação à viga maciça, mas somente para o módulo de ruptura à flexão. Essa eficiência foi expressa como a relação entre o momento de inércia real e o teórico. A emenda dentada não influenciou a elástica da viga laminada que apresentou o mesmo módulo de elasticidade da viga maciça. / The objective of the present study was to determine the shear strength in the bonding surface of special samples and to evaluate their impact on the geometric parameters of the finger, the tensile strength of the finger joints and the intrinsic flexural properties of simple and composed beams. The shear strength on the bonding surface was studied as a function of the inclination angle related to the wood fibers direction, from 0 to 90o in Eucalyptus grandis and Pinus elliottii species. Small inclination angles of the bonding surface performed very well in E. grandis samples and therefore it showed higher bonding efficiency when compared to P. Elliottii. The efficiency of the glued joints with different adhesives was determined by dividing the measured values by the shear strength of the solid wood. A simple theory relates the other known or simulated parameters of finger joints to the length of the finger. Using the values of the shear strength obtained experimentally with an inclination angle of 5o and the tensile strength of the solid wood of E. grandis finger length was calculated. Such theory, summarized in a unique equality, was developed to optimize the finger joints in terms of major acting efforts. The finger joints which had vertical and horizontal finger were tested in parallel traction to evaluate the maximum carrying load. The vertical profile had higher tensile strength and the difference was explained by the area of the glued joints in the finger. In the case of the vertical finger the area of glued joints was smaller and therefore had less impact when there was a defect in the finger or problem with the adhesion. Analyses of the modules of rupture revealed that rupture type 1 and type 5 or 6 contributed significantly to the reduction in the tensile strength of the finger joints. Rupture type 1 refers to failure due to causes intrinsic to the adhesive or to lack of pressure in the bonding process. Rupture type 5 or 6 refers to the presence of excessively weak points in the bar or to points of concentration of stress imposed by the clamps of the machine during the test. Such tensile strength must be characterized by the alternate presence of ruptures by mixed shear strength in the surface of the finger tip and the rupture by traction in the reduced section of the bar or in the base of the finger tip similar to a type 3 rupture. In flexural tests laminas which had a finger joint in the middle of the span ware tested as a beam subjected to tensile and compression normal stresses and also as a component of the laminated beam located in the traction edge. There were no significant differences between these two cases in any of the limiting states. The efficiency of the glued laminated beam with the finger joints located in the middle of the distance among points of support was very low in relationship to the efficiency of the solid wood, but only for the modulus of rupture in the flexure. This efficiency was expressed as the ratio of the moment of inertia to the theoretical moment. The finger joint did not influence the elasticity of the laminated beam since it had the same modulus of elasticity as the one found for solid wood.

A presente pesquisa teve como objetivo estudar a resistência ao cisalhamento no plano de cola de corpos de prova especiais e avaliar o seu impacto na geometria do dente, na resistência à tração de emendas dentadas e nas propriedades oriundas da flexão de vigas simples e compostas. A resistência ao cisalhamento no plano de cola foi estudada segundo o seu ângulo de inclinação em relação às fibras, desde 0 até 90o na madeira de Eucalyptus grandis e Pinus elliottii. O Eucalyptus grandis foi favorecido por pequenos ângulos de inclinação do plano de cola contrariamente ao que aconteceu com o Pinus elliottii e, portanto, apresentou maior eficiência na colagem, o que é desejável para viga laminada colada. Os valores obtidos foram divididos pela resistência ao cisalhamento da madeira maciça para encontrarem-se as eficiências das ligações coladas com vários adesivos. O valor da resistência ao cisalhamento no ângulo de 5° foi utilizado conjuntamente com a resistência à tração da madeira maciça de Eucalyptus grandis para calcular o comprimento do dente a partir de uma teoria simples que o relaciona aos demais parâmetros já conhecidos ou simulados da emenda dentada. Essa teoria, resumida numa única identidade, foi desenvolvida para otimizar a emenda em termos de seus esforços solicitantes majoritários.A emenda dentada foi testada à tração paralela em lâminas emendadas com dentes verticais e horizontais. O perfil vertical apresentou maior resistência à tração e a diferença foi explicada pela área de colagem individual do dente. No caso do dente vertical essa área era menor e, portanto, penalizava menos quando havia algum defeito no dente. Na análise dos tipos de ruptura apresentados verificou-se que a ruptura do tipo 1 que denota má colagem por causa intrínseca do adesivo ou falta de pressão na colagem e ruptura do tipo 5 ou 6 que evidenciam pontos excessivamente fracos da barra ligada ou pontos de concentração de tensões imposta pelas garras da máquina no ensaio de tração contribuíam significativamente para redução da resistência à tração da emenda dentada. Essa resistência deve ser caracterizada por ocorrência alternada de rupturas por cisalhamento misto na superfície do dente e ruptura por tração na seção reduzida da barra ou na raiz do dente como acontece na ruptura do tipo 3. Na flexão foram ensaiadas lâminas emendadas trabalhando como viga sujeita a tensões normais de tração e de compressão e lâminas emendadas trabalhando como componente de viga laminada posicionada na borda tracionada. Não houve diferença entre esses dois casos em qualquer dos estados limites. A eficiência da viga laminada colada com emenda no meio do vão foi muito baixa em relação à viga maciça, mas somente para o módulo de ruptura à flexão. Essa eficiência foi expressa como a relação entre o momento de inércia real e o teórico. A emenda dentada não influenciou a elástica da viga laminada que apresentou o mesmo módulo de elasticidade da viga maciça. / The objective of the present study was to determine the shear strength in the bonding surface of special samples and to evaluate their impact on the geometric parameters of the finger, the tensile strength of the finger joints and the intrinsic flexural properties of simple and composed beams. The shear strength on the bonding surface was studied as a function of the inclination angle related to the wood fibers direction, from 0 to 90o in Eucalyptus grandis and Pinus elliottii species. Small inclination angles of the bonding surface performed very well in E. grandis samples and therefore it showed higher bonding efficiency when compared to P. Elliottii. The efficiency of the glued joints with different adhesives was determined by dividing the measured values by the shear strength of the solid wood. A simple theory relates the other known or simulated parameters of finger joints to the length of the finger. Using the values of the shear strength obtained experimentally with an inclination angle of 5o and the tensile strength of the solid wood of E. grandis finger length was calculated. Such theory, summarized in a unique equality, was developed to optimize the finger joints in terms of major acting efforts. The finger joints which had vertical and horizontal finger were tested in parallel traction to evaluate the maximum carrying load. The vertical profile had higher tensile strength and the difference was explained by the area of the glued joints in the finger. In the case of the vertical finger the area of glued joints was smaller and therefore had less impact when there was a defect in the finger or problem with the adhesion. Analyses of the modules of rupture revealed that rupture type 1 and type 5 or 6 contributed significantly to the reduction in the tensile strength of the finger joints. Rupture type 1 refers to failure due to causes intrinsic to the adhesive or to lack of pressure in the bonding process. Rupture type 5 or 6 refers to the presence of excessively weak points in the bar or to points of concentration of stress imposed by the clamps of the machine during the test. Such tensile strength must be characterized by the alternate presence of ruptures by mixed shear strength in the surface of the finger tip and the rupture by traction in the reduced section of the bar or in the base of the finger tip similar to a type 3 rupture. In flexural tests laminas which had a finger joint in the middle of the span ware tested as a beam subjected to tensile and compression normal stresses and also as a component of the laminated beam located in the traction edge. There were no significant differences between these two cases in any of the limiting states. The efficiency of the glued laminated beam with the finger joints located in the middle of the distance among points of support was very low in relationship to the efficiency of the solid wood, but only for the modulus of rupture in the flexure. This efficiency was expressed as the ratio of the moment of inertia to the theoretical moment. The finger joint did not influence the elasticity of the laminated beam since it had the same modulus of elasticity as the one found for solid wood.

Les gènes IMA (Inhibitor of Meristem Activity) et MIF2 (MIni zinc Finger 2) codent des protéines de la famille des « MIni zinc Finger » impliquées dans le développement et la réponse hormonale. Leur fonction est intimement liée à leur capacité d’interaction avec des protéines partenaires au travers de leur unique domaine, un doigt de zinc non-canonique. La génération et la caractérisation de plantes gain- et perte-de-fonction pour ces gènes chez Solanum lycopersicum et Arabidopsis thaliana nous a permis de mettre en évidence leur homologie fonctionnelle dans le mécanisme d’arrêt de la prolifération des cellules souches méristématiques : la terminaison florale. Au cours du développement floral précoce, leur expression est induite par le facteur de transcription à MADS-BOX AGAMOUS. Les protéines codées sont capables d’intéragir avec le facteur de transcription à doigt de zinc C2H2 KNUCKLES (KNU) et de recruter le co-facteur TOPLESS (TPL) ainsi que l’HISTONE DEACETYLASE19 (HDA19) pour former un complexe multimérique impliqué dans le remodelage de la chromatine. Ce complexe intervient ensuite afin de réprimer l’expression du régulateur majeur de la prolifération des cellules souches méristématiques WUSCHEL. Les travaux qui ont suivi ont permis de montrer qu’au-delà de leur intervention dans ce processus, les protéines IMA et MIF2 sont impliquées d’une manière plus générale dans le développement floral et l’organogenèse. Leur fonction de protéine adaptatrice est sans doute permise par le désordre intrinsèque qui les caractérise et qui leur permettrait une spécificité d’interaction avec divers partenaires protéiques. / IMA (Inhibitor of Meristem Activity) and MIF2 (MIni zinc Finger 2) are two members of the MIni zinc Finger family (MIF) involved in the regulation of floral development and hormonal signaling pathways. Their ability to control physiological events is linked to their unique domain, a non canonical zinc finger, which confers to MIF the capacity to interact with other proteins. The characterization of gain- and loss-of-function lines for these genes in Solanum lycopersicum and Arabidopsis thaliana allowed us to unravel their functional homology in the termination of floral stem cell maintenance. During early floral development, IMA and MIF2 gene expression is induced by the MADS-Box transcription factor AGAMOUS. Then, IMA and MIF2 proteins recruit the C2H2 zinc finger KNUCKLES (KNU), in a transcriptional repressor complex together with TOPLESS (TPL) and HISTONE DEACETYLASE19 (HDA19). This complex binds to the WUSCHEL (WUS) locus leading to WUS repression via a chromatin deacetylation mechanism. Further work allowed us to demonstrate that IMA and MIF2 play a wider role in floral development and organogenesis. Their function as adaptor protein is probably linked to their intrinsic disorder leading to structural flexibility and interaction specificity with a large range of partners.

Η αποικοδόμηση των πρωτεϊνών είναι μια διαδικασία απαραίτητη για τη διατήρηση της ομοιόστασης του κυττάρου. Ένας από τους κύριους μηχανισμούς αποικοδόμησης των βραχύβιων πρωτεϊνών καθώς και όσων εμφανίζουν λανθασμένη αναδίπλωση, χωρεί μέσω του μονοπατιού ουβικιτίνης- πρωτεασώματος. Η ουβικιτινίωση είναι μια μετα-μεταφραστική διαδικασία, η οποία έγκειται στη σηματοδότηση των υποψήφιων για αποικοδόμηση πρωτεϊνών με ουβικιτίνη και περιλαμβάνει τρεις ενζυμικές ενεργότητες: Ε1 (εκκινητής ουβικιτίνης), Ε2(μεταφορέας ουβικιτίνης) και Ε3 (λιγάση ουβικιτίνης). Η πρωτεΐνη Arkadia (Rnf11) είναι μια Ε3 λιγάση ουβικιτίνης με συνολικό μήκος 994 αμινοξέα. Σε μοριακό επίπεδο, ενισχύει το TGF-β σηματοδοτικό μονοπάτι, διαμεσολαβώντας την εξαρτώμενη από το πρωτεάσωμα αποικοδόμηση των αρνητικών ρυθμιστών του, c-Ski και Sno-N. Η δραστικότητα Ε3 λιγάσης ουβικιτίνης εδράζεται στον C΄-τελικό RING-H2 τομέα, που σχηματίζεται από τα τελευταία 60 περίπου αμινοξέα της ακολουθίας. Η δομή και η σταθερότητα του RING τομέα εξαρτώνται από την πρόσδεση δύο ιόντων Zn μέσω ενός χαρακτηριστικού μοτίβου, που περιλαμβάνει 6 κυστεϊνικά και 2 ιστιδινικά κατάλοιπα. Στην προσπάθεια αποσαφήνισης της σχέσης δομής-δράσης της πρωτεΐνης Arkadia, ένα από τα κατάλοιπα που συναρμόζονται με Zn -συγκεκριμένα η His965- αντικαταστάθηκε από κυστεΐνη μέσω κατευθυνόμενης μεταλλαξιγένεσης. Η μετάλλαξη αυτή, με την οποία, ουσιαστικά, μετατρέψαμε τον RING-H2 σε RING-HC τομέα, μελετήθηκε με χρήση πολυπυρηνικής/πολυδιάστατης φασματοσκοπίας πυρηνικού μαγνητικού συντονισμού (NMR). H NMR δομή του RING-H2 τομέα της Η965C Arkadia επιλύθηκε σε υψηλή διακριτικότητα (tf=0.94±7.53*10-2, RMSD=0.75±0.20 και RMSD=1.45±0.24 για τα άτομα του πολυπεπτιδικού σκελετού και τα βαρέα άτομα αντίστοιχα) και αποκάλυψε μια ββαββ τοπολογία. Παράλληλα, πραγματοποιήθηκε μελέτη κινητικότητας, από την οποία προέκυψε ότι η εν λόγω μετάλλαξη υφίσταται ως μονομερές και διαθέτει έναν συμπαγή πυρήνα, που περικλείεται μεταξύ δύο ευκίνητων άκρων. / Protein degradation is necessary for the maintenance of cell homeostasis. A major mechanism for the degradation of short-lived as well as misfolded proteins involves the ubiquitin-proteasome pathway. Ubiquitination is a post translational modification, which targets the proteins to be degraded through the covalent attachment of a ubiquitin tag and consists of three enzyme activities: Ε1 (ubiquitin activator), E2 (ubiquitin carrier) and E3 (ubiquitin ligase). Arkadia (Rnf11) is a 994 amino acid protein, which acts as an E3 ubiquitin ligase. On a molecular level, Arkadia enhances TGF-β signaling by mediating the proteasome-dependent degradation of its negative regulators, c-Ski and Sno-N. Its E3 ubiquitin ligase activity lies on a C΄-terminal RING-H2 domain, formed by the last 60 residues. The structure as well as stability of the RING finger domain depend strongly on the binding of two zinc ions in a unique ΄΄cross-brace΄΄ arrangement through a defined motif of six cysteines and two histidines. Trying to elucidate the structure-activity relationship in the case of Arkadia, one of the amino acid ligands –specifically His965- was replaced by cysteine through site-directed mutagenesis. This particular mutation, which, in reality, transformed the RING-H2 to a RING-HC domain, was studied with the use of multinuclear/multidimensional nuclear magnetic resonance spectroscopy (NMR). The NMR solution structure of the H965 Arkadia RING-H2 domain was determined in high resolution (tf=0.94±7.53*10-2, RMSD=0.75±0.20 και RMSD=1.45±0.24 for backbone and heavy atoms respectively) and revealed a ββαββ topology. Furthermore, a mobility study was conducted with the following results: the mutated protein is not expected to form dimers and shows a compact core region including the four metal binding motifs flanked by two flexibly disordered termini.

本文旨從語料庫觀點來探討漢語手指謠之節奏、重音及手指動作之韻律結構。本研究建立了四千一百七十二行的語料庫，並詳細標記句法、虛詞、重音、音節和音拍等。本文發現手指謠節奏以陽性節奏 (Masculine Rhythm) 居多，音拍共享 (Beat-sharing) 常發生在虛詞 (F Categories) 和直接成分 (Immediate Constituents)，以符合陽性節奏的原則；透過詩行模組 (Metrical Template) 可衍生出不同的節律類型。另一方面，本研究認為重音在漢語中確實有存在之必要，藉由重音極大值 (Stress Maximum) 的落點，可解釋語料庫中不符詩感 (Metricality) 的謠行。本文最後將重心轉向手指謠的物理動作節律，手指動作因配合兒童年齡之認知及肢體發展，概分為0-2歲、2-4歲及4-6歲三大階段，越小的兒童吟誦手指謠之速度較慢，手指動作也相對簡單細微。本文發現手指動作範疇乃藉由語調片語 (Intonational Phrase) 來進行切割，語調片語須與「意義單位」 (Sense Unit) 彼此對整。本文亦從「優選理論」(Optimality Theory) 的角度來探討此三大研究議題，分別從「節律語法」和「流動制約」(Floating Constraints) 的觀念，藉由制約層級的重新排序來體現漢語手指謠的節律變化。 / This thesis takes great efforts in the topics about rhythm, stress and prosodic structures of the finger movements in Mandarin finger rhymes. This research established a corpus with a total of 4172 lines, each of which is coded with word categories, syntax tree, simplified tree, the number of syllables, the number of demibeats, and the stress. First of all, the masculine rhythm is the most common in the corpus. The beat-sharing, the strategy to obtain the masculine rhythm, is largely found in the F categories and the immediate constituents. The metrical template is also needed to yield the different rhythm types. Second, stress plays an important role in Maindarin Chinese. The judgement of the metricality strictly follows the metrical positions on which a stress maximum falls. The emphasis is finally put to the finger movements. The finger movements are respectively designed for the children who are 0-2, 2-4 and 4-6 years old, with the different developments in the cognition and physiology. When young children recite a metrical line, the speech rate is not as fast as other elder children and the finger movements are simple and subtle. The prosodic domains of the movements are defined from the perspective of IPs, and each IP is a sense unit (Selkirk 1984). Optimality Theory (Prince and Smolensky 1993, 2004) further covers these three research issues. The rhythm variations are vivid with the constraint re-rankings in terms of the metrics grammar and the floating constraints.

Convection set up in a fluid due to the presence of two components of differing diffusivities is known as double diffusive convection. Double diffusive convection is observed in nature, in oceans, in the formation of certain columnar rock structures and in stellar interiors. The major engineering applications of double diffusive convection are in the fields metallurgy and alloy solidification in casting processes. The two components may be any two substances which affect the density of the fluid, heat and salt being the pair found most commonly in nature. Depending upon the initial stratifications of the two components, double diffusive convection can be set up in either the diffusive mode or the finger mode. In this thesis, the linear stability of a double diffusive system prone to finger instability has been studied in the presence of temporally varying non-linear background profiles of temperature and salinity. The motivation for the present study is to bridge the gap between existing theories, which mainly concentrate on linear background profiles independent of time, on the one hand and experiments and numerical simulations, which have time dependent step-like non-linear background profiles, on the other. The general stability characteristics of a double diffusive system with step-like background profiles have been studied using the standard normal mode method. The background temperature and salinity profiles are assumed to follow the hyperbolic tangent function, since it has a step-like character. The sharpness of the step can be altered by changing a suitable parameter in the hyperbolic tangent function. It is found that changing the degree of non-linearity of the background profile of one of the components keeping the background profile of the other component linear affects the growth rate, Wave number and the form of the disturbances. In general, increasing the degree of nonlinearity of background salinity profile makes the system more unstable and results in a reduction in the vertical extent of the disturbances. On the other hand, increasing the degree of non-linearity of the background temperature profile with the salinity profile kept linear results in a reduction in the growth rate and increase in the wave number. The form of the disturbance may change due to enhanced modal competition between the gravest odd and even modes in this case. The method of normal modes inherently assumes that the background profiles of temperature and salinity are independent of time and hence, it cannot be used for studying the stability of systems with time varying background profiles. A pseudo-similarity method has been used to handle such background profiles. Initial steps of temperature and salinity diffuse according to the error function form, and hence, the case of error function background profiles has been studied in detail. Taking into account the time-dependence of background profiles has been shown to significantly change the wave number and the incipient flux ratio. The dependence of the critical wave number (kc) on the thermal Rayleigh number (RaT ) can be determined analytically and is found to change from kc ~ Ra T1/4 for linear background profiles to kc ~ Ra T1/3 for error function profiles. The region of instability in the Rp (density stability ratio) space is found to increase from 1 ≤ R ρ ≤ r−1 for linear background profiles to 1 ≤ Rρ < r−3/2 for error function background profiles, where T denotes the ratio of the diffusivity of the slower diffusing component to that of the faster diffusing one. A parametric study covering a wide range of parameter values has been carried out to determine the effect of the parameters density stability ratio (Rp), diffusivity ratio (ρ ) and Prandtl number (Pr) on the onset time, critical wavenumber and the incipient flux ratio. The wide range of governing parameters covered here is beyond the scope of experimental and numerical studies. Such a wide range can be covered by theoretical approaches alone. It has been shown that the time of onset of convection determines the thicknesses of the temperature and salinity boundary layers, which in turn determine the width of salt fingers. Finally, the theoretical predictions of salt finger widths have been shown to be in agreement with the results of two dimensional numerical simulations of thermohaline system.

No description available.

No description available.

Methoden zur direkten Detektion oder Anreicherung von doppelsträngiger DNA (dsDNA) bieten ein hohes Potential zum Einsatz in der molekularen Diagnostik. Bereits etablierte Methoden für die Nukleinsäure - Detektion (NAD) basieren in der Regel auf der Hybridisierung des komplementären Stranges gefolgt von der optischen Detektion oder enzymatischer Amplifikation. DNA - bindende oder organisierende Proteine (z.B. endogene Transkriptionsfaktoren) bieten im Kontrast zu den Hybridisierungsreaktionen eine überaus interessante Alternative um dsDNA direkt und zugleich spezifisch zu detektieren oder diese aus einem komplexen Gemisch heraus anzureichern. Im Rahmen der Entwicklung von neuartigen NAD - Assays zur direkten Detektion oder Anreicherung von Nukleinsäuren wurden vier DNA - bindende Proteine kloniert und in HEK293 und E. coli exprimiert. Der Cys2His2 - Zinkfinger (ZFD) vom humanen Transkriptionsfaktor Sp1 wurde mit MBP und 9×Lys - MBP fusioniert. Das MBP - Derivat 9×Lys - MBP ist eine erweiterte Variante mit neun aufeinanderfolgenden Lysinen im N - terminalen Bereich, welche eine regioselektive Immobilisierung ermöglichen soll. Der humane Sp3 - ZFD wurde mit EGFP fusioniert. Die Mitglieder der Sp - Familie binden spezifisch die Konsensussequenz 5’ - GGG GCG GGG - 3’ (GC - Box). Zusätzlich wurde die C - terminale DNA - bindende Domäne der E. coli DNA - Gyrase Untereinheit A (gyrA - CTD) ebenfalls mit MBP fusioniert. Die Domäne bindet spezifisch repetitive extragene Palindrome (REP), welche bislang nur auf bakteriellen Chromosomen vorkommen. Sämtliche MBP - Fusionsproteine liegen nach der Expression löslich vor und konnten über eine native Strategie aufgereinigt werden. Transiente Transfektionsexperimente in HEK293 zeigten einen destabilisierenden Effekt der Sp3 - ZFD und eine massive einhergehende Degradierung des EGFP - Fusionsproteins nach 120 h. Die Analyse der mRNA - Integrität nach Transfektion des Expressionsplasmids, sowie zellbiologische und proteinbiochemische Untersuchungen mit Durchflusszytometrie bzw. Western Blots deuten auf eine posttranslationale Modulierung von EGFP - Sp3 hin. Um die Hypothese der proteasomalen Degradierung von EGFP - Sp3 zu belegen, wurden transfizierte HEK293 mit dem reversiblen Proteasominhibitor MG132 behandelt. In Gegenwart von 1 µM MG132 konnte das zytosolische Fusionsprotein stabilisiert werden. Die hier präsentierten Daten offenbaren die humane Sp3 - ZFD als ein neues Substrat für das 26S - Proteasom. Lediglich die SUMOylierung von Wildtyp - Sp3 im Bereich der inhibitorischen Domäne (ID) ist bislang beschrieben worden. Die Funktionalität, Affinität und kinetische Parameter der mit MBP fusionierten Sp1 - ZFD und gyrA - CTD wurden anhand von Oberflächenplasmonresonanz (BIAcore) bzw. EMSAs analysiert. Sämtliche gewonnenen MBP - Fusionsproteine sind funktionell und interagieren mit dsDNA. Fusionsproteine mit Sp1 - Domäne zeigten in EMSAs ebenso eine Bindung an unspezifische dsDNA. In sensitiveren BIAcore - Assays mit immobilisierter dsDNA wurden (um den Faktor 2) geringere Assoziations (ka) - und Dissoziationsraten (kd) von MBP - Sp1 ermittelt, wenn bestimmte Basen innerhalb der GC - Box ausgetauscht wurden. Die Affinität (Kd) von MBP - Sp1 mit 4×10 - 9 M zur GC - Box und deren Derivate ist vergleichbar mit der Kd von nativem Sp1. Die EMSA - Experimente für MBP - gyrA zeigen eine deutliche Präferenz zum spezifischen dsDNA - Oligo in Gegenwart von humaner gDNA, eine interessante Eigenschaft die durchaus zur Anwendung in einem Assay zur Anreicherung von bakterieller DNA dienen kann. Nach der vorausgehenden Charakterisierung der MBP - Fusionsproteine wurden diese auf verschiedenen gängigen festen und semifesten Substraten über physische Adsorption, kovalent oder Affinität immobilisiert um das Konzept der direkten Detektion von dsDNA mit funktionellen Proteinen als neuartige Komponente in NAD - Assays umzusetzen. Lediglich MBP - Sp1 zeigte auf Glas und Polystyren - Mikrotiterplatten nach kovalenter oder adsorptiver Immobilisierung eine ausgeprägte Funktionalität hinsichtlich der Bindung von dsDNA. Die Immobilisierung von 9×Lys - MBP - Sp1 über identische Strategien führten zum massiven Verlust der ZFD - Funktion. Aus dieser Datenlage heraus wurde erfolgreich ein simples Lumineszenz - basiertes Mikrotiterplatten - Assay mit MBP - Sp1 entwickelt um PCR - Amplikons direkt aus einer analytischen PCR auf gDNA von S. aureus, welche die GC - Box beinhalten, nachzuweisen. Das spezifische Amplikon konnte mittels des simplen Assays in Gegenwart von 100fachem Überschuss an humaner gDNA nachgewiesen werden. Mit einem höheren Anteil an humaner gDNA wurde die PCR massiv inhibiert, ein negativer Effekt der bislang im Bereich der diagnostischen NAD - Assays nicht optimal adressiert wurde. Die magnetische Separation von bakterieller und humaner gDNA wurde dazu mit MBP - gyrA umgesetzt. Zunächst erfolgte die regioselektive Immobilisierung von MBP - gyrA auf Protein A - funktionalisierte magnetische Nanopartikel mittels MBP - Antikörper, wodurch die Funktionalität hinsichtlich der Bindung von dsDNA gewährleistet werden konnte. Dieses System eignet sich insbesondere für die Separation von bakterieller DNA (E. coli oder S. aureus) aus einem komplexen Gemisch mit bis zu 100fachem Überschuss an humaner gDNA. Die Kombination von MBP - gyrA - basierter magnetischer Separation mit NAD - Assays könnte deren Sensitivität signifikant erhöhen. Durch simple Verfahrensweise bietet das System einen wesentlichen Beitrag zur Verringerung des zeitlichen Aufwands für die Generierung therapierelevanter Resultate. / Methods for direct detection or enrichment of double - stranded DNA (dsDNA) possess tremendous potential for use in molecular diagnostics. Already established methods for nucleic acid detection (NAD) are generally based on the hybridization of two complementary strands followed by optical detection or enzymatic amplification. In contrast, DNA - binding or organizing proteins (e.g. endogenous transcriptions factors) are able to read the sequence information directly from dsDNA without prior denaturation of the double strand and subsequent hybridization. In order to develop novel NAD assays or assays for sample preparation, four artificial DNA - binding proteins were cloned, expressed and purified in HEK293 cells or E. coli. The Cys2His2 zinc finger domains (ZFD) from human Sp1 were fused to maltose binding protein (MBP) and its derivate 9×Lys - MBP, an extended variant with nine successive lysine residues in the N - terminal region of the protein to facilitate site - directed immobilization. The human Sp3 - ZFD was fused to green fluorescent protein (EGFP). The family of Sp - transcription factors was known to bind specifically the consensus sequence 5\' - GGG GCG GGG - 3 \'(GC - box). Moreover, the C - terminal DNA - binding domain of E. coli DNA Gyrase subunit A (gyrA - CTD) was fused to MBP. The CTD binds specifically repetitive extragenic palindromes (REP), which were only found on prokaryotic chromosomes. All MBP fusion proteins were soluble after expression and could be purified to homogeneity. Surprisingly, transient transfection experiments in HEK293 revealed a destabilizing effect of the Sp3 - ZFD accompanied by massive degradation of the EGFP fusion protein after 120 h post transfection. Analysis of mRNA integrity in combination with western blots indicates a posttranslational modulation of EGFP - Sp3. To confirm the hypothesis of proteasomal degradation of EGFP - Sp3, transfected cells were treated with the reversible proteasome inhibitor MG132. In the presence of 1µM MG132 the fusion protein could be stabilized. Taken together, the data presented here identified the human Sp3 - CTD as a new substrate for the 26S proteasome. Only SUMOylation of wild type human Sp3 within the inhibitory domain (ID) has been described so far. Initial EMSA experiments showed that purified MBP - ZFD fusion proteins were functional in terms of interacting with dsDNA containing the specific sequence motiv. However, all proteins bound to unspecific dsDNA as well. Therefore MBP - Sp1 was subjected to BIAcore analysis to determine the rate constants for association ka, dissociation kd and the dissociation constant Kd of the GC - Box - Protein complex as well as mutants of the GC - Box. The determined Kd (4 × 10 - 9 M) for MBP - Sp1 associated with GC - box or its derivatives were found to be comparable with the Kd of native Sp1, however the rate constants were reduced 2 fold in presence of the modified GC - boxes. EMSA experiments with MBP - gyrA revealed functionality and a clear preference for specific dsDNA in the presence of unspecific human genomic DNA (gDNA). After preliminary functional characterization, MBP fusion proteins were immobilized by physical adsorption, covalent or by affinity on various solid substrates or nanoscaled magnetic beads to implement the concept of direct detection of dsDNA or specific enrichment of bacterial DNA, respectively. MBP - Sp1 remains functional after adsorptive or covalent immobilization on different chemical modified glas surfaces. 9×Lys - MBP - Sp1 shows significantly reduced functionality after immobilization on the same glas substrates by similar strategies. Moreover, a simple NAD - assay with adsorptive immobilized MBP - Sp1 on polystyrene in microtiter format was established for direct detection of GC - boxes within PCR - products from S. aureus gDNA. By using the assay, specific PCR - products could be detected in presence up to 100 - fold excess of human gDNA in relation to 10 ng bacterial DNA. Separation of bacterial DNA from human DNA from clinical samples may have an important impact on downstream applications, involving NAD assays. To address this often underestimated technical problem, a new functional protein MBP - gyrA was introduced to overcome some limitations of already established methods. MBP - gyrA was site - directed coupled on nanoscaled magnetic beads by affinity. This system enabled the fast and specific separation of gDNA of E. coli or S.aureus from a huge background of human gDNA. The combination of MBP - gyrA - based magnetic separation with NAD assays could significantly increase the sensitivity and shorten the time for initiation of effective treatment.