Die Etablierung der Transkription von kompletten Genen auf planaren Oberflächen soll eine Verbindung zwischen der Mikroarraytechnologie und der Transkriptomforschung herstellen. Darüber hinaus kann mit diesem Verfahren ein Brückenschlag zwischen der Synthese der Gene und ihrer kodierenden Proteine auf einer Oberfläche erfolgen. Alle transkribierten RNAs wurden mittels RT-PCR in cDNA umgeschrieben und in einer genspezifischen PCR amplifiziert. Die PCR-Produkte wurden hierfür entweder per Hand oder maschinell auf die Oberfläche transferiert. Über eine Oberflächen-PCR war es möglich, die Gensequenz des Reportergens EGFP direkt auf der Oberfläche zu synthetisieren und anschließend zu transkribieren. Somit war eine Transkription mit weniger als 1 ng an Matrize möglich. Der Vorteil einer Oberflächen-Transkription gegenüber der in Lösung liegt in der mehrfachen Verwendung der immobilisierten Matrize, wie sie in dieser Arbeit dreimal erfolgreich absolviert wurde. Die Oberflächen-Translation des EGFP-Gens konnte ebenfalls zweimal an einer immobilisierten Matrize gezeigt werden, wobei Zweifel über eine echte Festphasen-Translation nicht ausgeräumt werden konnten. Zusammenfassend kann festgestellt werden, dass die Transkription und Translation von immobilisierten Gensequenzen auf planaren Oberflächen möglich ist, wofür die linearen Matrizen direkt auf der Oberfläche synthetisiert werden können. / In vitro mRNA synthesis and in vitro translation are of great interest for biochemical and molecular biological basic research, and also for biotechnology and other applications. Solid phase coupled synthesis is very useful for the development of high throughput procedures to elucidate and manipulate gene products. An artificial gene was constructed combining the T7 promoter and terminator with the EGFP-gene from the plasmid pEGFP. The functionality of the construct was shown by in vitro translation. The gene-construct was immobilised on a planar glass surface. The transcription was performed on the immobilised gene and mRNA was determined by RT-PCR. These results demonstrate that the complete gene is transcribed from the covalently coupled PCR product. Thus, it is possible to transfer a standard transcription technique onto an On-chip reaction. The direct PCR amplification of transcriptionable sequences of EGFP bound on surfaces was successfully used for solid phase transcription. Successful transcriptions were also performed at least to 1 ng of used template. The RNA synthesis was also successful in the second and third reaction on the same slide as observed by signals after RT-PCR. It seems to be possible to transfer the translation of reportergenes in a solid phase coupled synthesis, too. For further integration of cellular procedures on a chip, the cell-free RNA synthesis on immobilised templates is an crucial technical hurdle to conquer. Major advantages of using immobilised templates for transcription are, low risk of contamination occuring in solution, and no necessity of further purification steps for downstream applications of the RNA product.

Da interação da actina com a miosina resulta a contração muscular. Esta interação é controlada pela concentração de íons cálcio, que atuam sobre um sistema regulatório associado ao filamento de actina. O sistema regulatório é composto de uma molécula de tropomiosina e um complexo protéico composto de três polipeptídeos chamado troponina. Uma das subunidades, troponina C, e o receptor de ions cálcio; outra, troponina T, liga-se à tropomiosina; e a terceira, troponina I, é a responsável pela inibição da atividade ATPásica da actomiosina. Esta dissertação descreve o desenvolvimento do processo de expressão da troponina I em bactéria, sua purificação e caracterização. Partindo de um cDNA de troponina I músculo esquelético de galinha, procedemos à construção de um vetor de expressão da proteína em E.coli com o sistema pET. Inicialmente construímos um vetor de expressão capaz de produzir a troponina I fundida a 18 aminoácidos. A proteína foi purificada e caracterizada, comportando-se de forma semelhante à troponina I selvagem. Numa segunda etapa, foram feitas mutações sítio-dirigidas para a construção de um novo sítio de restrição que possibilitou construir um vetor de expressão da troponina I a partir de sua metionina inicial. Entretanto não foi obtida expressão com esta construção. Para solucionar o problema, foram feitas novas mutações que alteraram os codons AGG presentes nos 25 primeiros codons do gene da proteína por codons CGT, sem alterar o aminoácido codificado. Nesta construção final foi obtida a expressão da proteína sem fusão, que purificada e caracterizada, comporta-se de modo idêntico à troponina I selvagem. / Muscle contraction results from the interaction of myosin with actin and this interaction is controlled by the calcium ion concentration which acts on the regulatory system associated with the actin filaments. This regulatory system comprises one tropomyosin molecule and a complex composed of three polypeptide subunits. One of this subunits, troponin C binds calcium ions; another, troponin T, binds to tropomyosin while the third, troponin I, inhibits the ATPase activity of actomyosin. This dissertation describes the development of the methodology to express troponin I in bacteria and its subsequent purification and characterization. Starting with a troponin I cDNA from chicken skeletal muscle, a pET expression vector was constructed. The initial vector resulted the expression of a troponin I fusion protein having an additional 18 amino acids. This protein was purified and characterized and found to behave like wild type troponin I. Subsequently, employing site-directed mutagenesis, a new vector was made which allowed the expression of the protein starting at its initial methionine codon and lacking the extra amino acids. However, no expression was achieved using this vector and, to circumvent this, further mutations were introduced in the first 25 codons which substituted AGG for CGT without altering the amino acid sequence. This final construct permitted the expression of a non fusion troponin I which, after purification and characterization, was found to behave identically to the wild type protein.

Thema dieser Arbeit ist die evolutive Optimierung der T7-RNA-Polymerase. Zur Stabilisierung technischer oder therapeutischer RNA-Moleküle gegenüber RNAsen wäre es wünschenswert eine RNA-Polymerase zu generieren, welche RNA vollständig aus 2’- modifizierten Nucleotiden synthetisieren kann. Zu diesem Zweck wurde ein kombiniertes Selektions- und Screeningverfahren zur Durchmusterung von Variantenbibliotheken der T7- RNA-Polymerase hinsichtlich des Einbaus von 2’-Methoxy-modifizierten Nucleotiden in RNA entwickelt. Es wurden ein gut handhabbarer, cis-regulierter Expressionsvektor sowie ein Selektionsplasmid erzeugt, die zusammen in E. coli ein in-vivo-Selektionssystem bilden, mit dessen Hilfe man Zellen, welche T7-RNA-Polymerase-Aktivität zeigen anhand ihrer grünen Fluoreszenz identifizieren konnte. Durch error-prone PCR wurden Mutantenbibliotheken generiert, und diese in das Selektionssystem eingesetzt. So konnte die Anzahl der potentiell zu testenden Varianten erheblich gesenkt werden. Zur Bestimmung der T7-RNA-Polymerase- Aktivität mit 2’-Methoxy-modifizierten Nucleotiden wurde ein Fluoreszenz-basierendes Assay etabliert. Dieses Assay, das nicht mit radioaktiv-markierten Nucleotiden arbeitete und keinen gelelektrophoretischen Separationsschritt benötigte, konnte in allen Schritten zur parallelen Bearbeitung von 96 Proben in einem Mikrotiterplatten-Format angepasst werden, so dass es prinzipiell hochdurchsatzfähig war und sich zum Screening umfangreicher Variantenbibliotheken eignete. Die Assay-Reaktion kann dabei auch unkompliziert auf ein Screening von RNA- oder DNA-Polymerase-Bibliotheken hinsichtlich anderer Eigenschaften der Polymerase-Aktivität übertragen werden.

Dans ce travail, nous proposons un nouvel appareillage, destiné à aider à la détermination du mécanisme de certaines protéines. Cet outil, qui combine un appareil de pinces magnétiques, et un microscope de fluorescence en ondes évanescentes, a été conçu pour permettre à la fois la manipulation mécanique et l'observation de l'activité d'ADN translocases à l'échelle de la molécule unique. Nous présentons d'abord ici la conception, la réalisation et l'expérimentation de ce montage. Nous montrons que, d'une part, il se compare favorablement à ses composants séparés (pinces magnétiques et microscope de fluorescence), et que d'autre part leur réunion dans un appareil unique permet d'obtenir des résultats d'un type nouveau. Nous avons orienté l'étude des ADN translocases avec cet appareil sur l'exemple de deux protéines : le moteur FtsK de Escherichia coli et l'ARN Polymérase de T7. Nous détaillons dans cette étude les questions importantes encore en suspens concernant le mécanisme et présentons les expériences que nous avons envisagées pour y répondre. Nous relatons ensuite également la difficulté que nous avons rencontrée à obtenir des substrats protéiques adaptés aux expériences que nous avons envisagées, et les solutions que nous avons mises en oeuvre pour y remédier. Enn, nous analysons les résultats obtenus dans des expériences en volume ou en pinces magnétiques seules, qui ont permis de mettre en valeur de nouveaux comportements et de préparer la réalisation de nouvelles expériences sur notre montage combiné.

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Civil and Environmental Engineering, 1998. / Includes bibliographical references (p. 181-184) and index. / This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / This study develops a model of a person's day activity schedule that can be used to forecast urban travel demand. It is motivated by the notion that travel outcomes are part of an activity scheduling decision, and uses discrete choice models to address the basic modeling problem-capturing decision interactions among the many choice dimensions of the immense activity schedule choice set. An integrated system of choice models represents a person's day activity schedule as an activity pattern and a set of tours. A pattern model identifies purposes, priorities and structure of the day's activities and travel. Conditional tour models describe timing, location and access mode of on-tour activities. The system captures trade-offs people consider, when faced with space and time constraints, among patterns that can include at-home and on-tour activities, multiple tours and trip chaining. It captures sensitivity of pattern choice to activity and travel conditions through a measure of expected tour utility arising from the tour models. When travel and activity conditions change, the relative attractiveness of patterns changes because expected tour utility changes differently for different patterns. An empirical implementation of the model system for Portland, Oregon, establishes the feasibility of specifying, estimating and using it for forecasting. Estimation results match a priori expectations of lifestyle effects on activity selection, including those of (a) household structure and role, such as for females with children, (b) capabilities, such as income, and (c) activity commitments, such as usual work levels. / (cont.) They also confirm the significance of activity and travel accessibility in pattern choice. Application of the model with road pricing and other policies demonstrates its lifestyle effects and how it captures pattern shifting-with accompanying travel changes-that goes undetected by more narrowly focused trip-based and tour-based systems. Although the model has not yet been validated in before-and-after prediction studies, this study gives strong evidence of its behavioral soundness, current practicality, potential to generate cost-effective predictions superior to those of the best existing systems, and potential for enhanced implementations as computing technology advances. / by John L. Bowman. / Ph.D.

<p>Borane (BH<sub>3<sub>) chemistry offers unique chemical characteristics that enable boranophosphate (BP) oligonucleotides with potential to enhance RNA therapeutic applications such as RNA interference (RNAi) and RNA aptamers. Further, BP nucleotides are substrates for RNA polymerases which allow the enzymatic synthesis of stereoregular boranophosphate (BP)-RNA molecules of different lengths and properties. We expect that these BP-RNAs will interact in a novel way with the desired target molecules because they can coordinate with a diverse array of ligand sites in proteins or other RNA molecules. This is due to the distinct hydrophobicity, sterospecificity, and polarity properties imparted by the phosphorus-boron (P-B) chemical bond compared to the natural phosphorus-oxygen (P-O) bond. </p><p>The object of this dissertation is to explore the therapeutic applications of the BP-RNA such as siRNA, RNA aptamers, and in addition investigate the immunogenicity of this modification. We used mouse cells to determine if BP-RNA would activate toll-like receptor (TLR 7), which is involved in innate immune response to foreign single stranded RNA (ssRNA). This response is undesired when applied to oligonucleotide therapeutics such as siRNA and RNA aptamers. In terms of RNAi, it would be an advantage to have low immunogenicity and high downregulation activity by the siRNA. To determine the innate immune activation of the BP-RNA through the TLR 7 we used a known activator, the human immunodeficiency virus (HIV) derived single-stranded RNA (ssRNA40) and measured the production of cytokines as a function of the number of modified BP-linkages. The production of cytokines IL-6 and TNF&#945; was quantified after the boranophosphate (BP), phosphorothioate (PS) or natural ssRNA40 were transfected into murine macrophage Raw264.7 cells. Natural and phosphorothioate RNA (PS-RNA) have been shown to be activators of TLR 7 receptors. In contrast, we found that fully modified BP- ssRNA40 did not activate TLR 7. This is relevant in oligonucleotide applications such as siRNA and RNA aptamers where off-target effects such as immune activation after administration are not desired. </p><p>Subsequently, the low immune activation would be an advantage when coupled to RNAi activity of the oligonucleotide. Thus, we explored whether BP modified siRNA molecules would modulate gene expression and if there was an effect on downregulation activity when increasing the number of BH3 modifications on the phosphate backbone. Our therapeutic model was the multi-drug resistance 1 (MDR1) gene that expresses P-glycoprotein (P-gp), which has been notoriously difficult to modulate. The aberrant regulation of genes such as MDR1 in cancer cells are a major cause of chemotherapeutic treatment failure against human cancers. Hence, controlling the expression of cancer genes with antisense technology is a possible cancer therapy. Specifically, correcting the overexpression of p-glycoprotein using modified siRNAs that target and degrade the P-glycoprotein mRNA produced by the MDR1 gene. We found that there is a reduction of siRNA activity with an increasing number of BP-modifications. It appears that there is a fine balance between lack of immune response and gene downregulation when applied to BP-siRNA. </p><p>Finally, we compared the enrichment during the Systematic Evolution of Ligands by EXponential enrichment (SELEX) method of two libraries, one BP-RNA (U&#945;B) compared to a doubly-modified RNA (2'FC & U&#945;B), against a human thrombin. Aptamers modulate protein activity and interfere with protein signaling by binding to the desired protein with high affinity and specificity leading to their use in therapeutic applications where protein activity needs to be controlled or it is anomalous. In the case of blood coagulation, thrombin plays a central role in coagulation signaling cascade and it is a good target to use to control blood coagulation in clinical settings. We attempted to optimize the selection of BP- RNA aptamers through 4-8 rounds of SELEX against the protein thrombin. We found that the selection conditions were not optimal for BP-RNA SELEX possibly due to non-specific binding to a bovine serum albumin (BSA) in the selection buffer.</p> / Dissertation

National cultural and institutional factors and the influence of the parent company affect the ways in which Multinational Corporation subsidiaries train and develop their staff. However, existing studies have not compared training and development using a comprehensive range of comparative factors. Thus the key determinants of the organization, planning and conduct of training and development are not identified or explained. This study develops a comprehensive series of factors that can be used to compare training and development in the British and French subsidiaries of a Swedish Multinational Corporation. The hypothetical implications of national culture, the national institutional context and the national origin of the parent company are identified. It is expected that national cultural and institutional factors will have a greater influence upon training and development than the actions of the parent company, and that cultural factors will be more influential than institutional factors. The hypotheses are tested in a study that involves interviewing company staff and training providers in Britain, France and Sweden. A range of company documents is also gathered. Analysis of the data reveals that the parent company has virtually no influence on the training and development actions of the subsidiaries, although there are signs that this ‘lack of management’ may be under review. Further analysis reveals that national culture is the key determinant of the ways in which training is conducted in the subsidiaries. However, cultural explanations for the ways in which the subsidiaries organize and plan training and development are rejected. The competitive market environment that the subsidiaries operate in, especially the ability (or not) to gain market share seems to be the key determinant of the organization and planning of training and development in the subsidiaries. It is concluded that this is a significant finding which should be explored in future studies

Leo VI (886-912) is an emperor who has suffered from a hostile and inadequate press. He has been portrayed as a weak and careless emperor, known mainly for his dubious parentage and marital exploits. This thesis questions these popular perceptions of Leo, and attempts to present a more realistic account of the emperor and the politics of his age. The aspects of the reign tackled focus on essential elements of Leo's life and rule, presented in a rough chronological framework, and the themes of personal relationships and political ideologies are recurrent. Chapter One examines Leo's relationship with Basil I and his attitude to his Macedonian heritage. Chapter Two considers the fate of the monumental figure of Photios at the emperor's hands. Chapter Three deals with the position and role of the 'all powerful' Stylianos Zaoutzes during the first half of the reign. Chapter Four ponders the origin and meaning of Leo's 'wise' epithet. Chapter Five focuses on the emperor's four marriages. Chapter Six turns to the course of foreign affairs during the reign, concentrating on Bulgaria and the Arab navy, and considers the emperor's attitude towards these military problems. Chapter Seven examines the emperor's relationship with his senatorial officials, focusing on two distinct groups, eunuchs and the generals who originated from families of the eastern frontier. Finally Chapter Eight addresses the tense relationship that existed between Leo and his brother and co-emperor Alexander. What emerges from a consideration of these aspects of Leo and his reign is that this is an emperor who does not deserve the popular perceptions that still persist about him. He was an emperor who forged a 'new' and distinctive imperial style, a style that should not deceive us; he may have been literate, sedentary and city-based, but he was also forceful, strong-willed and conscientious.

In order to monitor the T7 replisome fate upon encountering abasic lesion, I optimized a single molecule flow stretching assay where the replisome encounters either abasic site or undamaged site inserted at 3.5 kilobases from the replication fork. The obtained events were categorized into three groups; bypass, restart and permanent stop. The results showed 52% bypass, 39% pause and 9% stop upon encountering the abasic lesion. The pause duration in the restart events was found to be ten times longer than the undamaged one. Moreover, an ensemble experiment was performed, and the results were slightly consistent with regard to the bypass percentage (70%) but the stoppage percentage was significantly higher in the ensemble replication reaction (30%). Further investigations were made and it was found that the rate of the T7 replisome increases after bypassing the abasic lesion. To inquire more about this rate switch and the difference between the single molecule and ensemble results, another unwinding experiment was performed where only gp4 (helicase) was used from the replisome. Interestingly, the rate of DNA unwinding by gp4 was similar to the rate observed after the replisome bypasses the lesion. We hypothesize that the polymerase is stalled at the abasic site and its interaction with the helicase is lost. Consequently, the helicase and the polymerase will uncouple where the helicase continues unwinding the DNA to result in a higher observed rate after bypassing the abasic site. Additional studies will be performed in the future to directly observe the helicase and polymerase uncoupling upon encountering the lesion.

A method is described for the rapid purification of RNA polymerase holoenzyme from small amounts of Escherichia coli cells. Chromatography of a crude extract on a single-stranded DNA agarose column followed by gell filtration chromatography gave 95% pure holoenzyme. The enzyme kinetic characteristics on T7 DNA identical to those of RNA polymerase purified by other more laborious procedures.