Members of multi-specific DNA-binding protein families have evolved to specifically recognize diverse DNA site sequences. This dissertation presents evidence that the Cro protein family of helix-turn-helix transcription factors from lambdoid bacteriophages may share a conserved, limited "code" that partially governs evolution of their binding specificity. A bioinformatic study revealed six conserved sequence correlations between residues at three positions in Cro recognition helices and three base-pairs in putative cognate DNA consensus half-sites (Chapter 2). Three of these pairings correspond to sequence-specific contacts observed at the binding interface of lambda Cro and consensus operator DNA in a previously available co-crystal structure (Albright and Matthews, 1998a). In vitro mutagenesis and functional characterization was used to validate the proposed "code" (Chapter 3). Two out of three "coding" combinations acted as specificity switches in lambda Cro, though variant proteins displayed reduced binding specificity for their predicted target DNA sites. Two crystal structures of a lambda Cro variant are presented in Chapter 4, which provide insight into lambda Cro dimer flexibility. Additionally, a co-crystal structure of N15 Cro bound to consensus site DNA was determined which contains two coding residue pairs at the binding interface (Chapter 5), and a crystal structure of Xfasa1 Cro that enables future investigations into Cro functional evolution (Chapter 6). Although there are several caveats, the data are consistent with a model in which Cro proteins may indeed have evolved new binding specificities in part through simple mutations at their binding interfaces that follow a simple set of evolutionarily conserved "coding" rules. The structural and functional diversity of Cro proteins provides an exciting venue for future research into their evolution.

Diss.--Kulturgeschichte und Kulturkunde--Universität Hamburg, 1994. / Bibliogr. p. 311-327.

Natural proteins can evolve new three-dimensional structures through mutations in their amino acid sequence. For protein families that exhibit such structural diversity, a major challenge is to understand the scope and nature of structure variation and its relationship to sequence evolution. The Cordes laboratory has begun using transitive homology-based methods to identify, target and structurally characterize natural sequences intermediate between pairs of proteins with distant sequence similarity and different structures. As a proof of principle, this dissertation describes structural studies of two proteins in different families as separate case studies, one involving secondary structure evolution and the other involving topological rearrangement. In the first case, crystallography was applied to solve the structure of a sequence intermediate identified through transitive homology analysis of the Cro transcription regulator family. Comparison with another member resulted in finding two proteins with significant sequence similarity yet different secondary structure compositions and folds. In the second case, transitive homology analysis was applied to look at two members of the insect salivary lipocalins, one with the canonical sequential all-antiparallel β-barrel topology, and another with a unique strand-swapped topology. Three sequence intermediate members were found that each have direct sequence similarity to both topologically distinct relatives. Targeting these sequence intermediate members for structural characterization by NMR led to assignment of the canonical lipocalin topology for one intermediate. The results from these two cases indicate that structurally diverse families may contain members with similar sequences but different folds. As such, transitive homology mapping offers a method to identify and target those members for structural characterization.

Abstract Bio-technology has been viewed as the ¡§star¡¨ industry in the 21st century. To entrepreneurs around the world bio-technology has offered an enormous opportunity for development. Recently partly because of the encouragement of government incentive programme a large number of bio-tech related firms have been established. In Taiwan these firms are associated with several characteristics: small but innovative; high growth; highly active; technology-intensive; high R& D spending; low on manufacturing; lacking attention to intangible asset and so on. By synthesizing the literature review and intensive case studying, this study has concluded that the bio-tech firms in Taiwan have faced a number of key barriers including lacking of capital and capital trapping, lacking confidence and infrastructure, lacking attention to R&D. This study has adopted, in conjunction with those developed by Chen (year) and Tan (year), the Michael Porter¡¦s diamond model to develop its research framework (Porter, year). This study has also utilised the SWOT and the five force model analysis method in its framework. This study has analysed two firms from each of the following industrial sectors: The research framework was proposed in light of intensive literature review. The research framework was examined by the data collected from both primary and secondary research. The main data collection methods employed were personal interviews with experts specialised in bio-technology and some secondary information. The key findings of this study include: (1) Though some traditional bio-tech industrial sectors are already saturated, some bio-technology related sectors are still emerging and developing. (2) It is found that the most important factors influencing CMO is the transaction cost, followed by resource dependence. (3) Transaction cost is negatively related to CMO while resource dependence is positively associated with CMO. The degree of satisfaction of CMO is also positively related to the degree of CMO. (4) Consistent with existing literature.The research concerning CMO needs to be explored from three aspects: economic costing, operational strategy and social costing. (5) Strategic alliance is widely evidenced in the management, for example, in the areas of research and development and marketing. In the future bio-technology firms are suggested to well utilise the capabilities of major research centres in order to overcome the barriers caused by the inadequacy of institutional laws and lacking of research funds. Key word:Biotech, Biotechnolog, Biotechnolog industry, CMO, CRO, SWOT Analysis¡C

Notre travail porte sur les représentations des élèves de CE2, CM1 et CM2 sur la préhistoire et Cro-Magnon en articulant des éclairages propres à la didactique de l’histoire (modèle intermédiaire d’appropriation de l’histoire) et à la psychologie sociale (théorie des représentations sociales). Nous sommes partis du postulat que les savoirs de l’histoire scolaire étaient simplifiés à l’extrême, emplis de sens commun et parfois en contradiction avec les avancées récentes de la recherche. Nous avons émis l’hypothèse que la représentation des élèves sur la préhistoire était particulièrement unifiée, sous l’influence des représentations sociales historiques et socioculturelles, malgré l’intervention de l’enseignement. Après avoir présenté le cadre théorique, nous avons réalisé une étude épistémologique historique sur la préhistoire et Cro-Magnon avec une analyse des représentations historiques à partir d’une étude bibliographique, une synthèse des analyses récentes sur les représentations de la préhistoire et une synthèse des données scientifiques actuelles sur la préhistoire. Les analyses issues de l’objet d’étude ont permis d’identifier les principaux obstacles épistémologiques et les représentations sous-jacentes. Nous avons ensuite procédé à l’analyse de données empiriques que nous avons recueillies sous plusieurs formes : des questionnaires d’évocations hiérarchisées et des questionnaires de caractérisation. Une étude plus standard à l’aide d’un questionnaire classique a complété le dispositif : ce questionnaire, distribué avant, pendant et après le cours de préhistoire pour le niveau CE2, a permis à travers une étude diachronique de mesurer l’influence de l’enseignement sur les représentations des élèves. Les résultats ont été confrontés à ceux de l’objet d’étude et ont rendu possible l’identification de représentations ainsi que les principes organisateurs de prise de position c’est-à-dire leur ancrage socioculturel. Nous avons montré que le sens attribué à l’objet de représentation sociale de la Préhistoire est le résultat d’une convergence de différents facteurs dont finalement l’enseignement n’est qu’un élément périphérique voire marginal. L’influence majeure provient des déterminants historiques et socioculturels. Le savoir scolaire de sens commun élaboré à l’issue de l’intervention de l’enseignant était proche à bien des égards du savoir prescrit parce que ce dernier est aussi, en grande partie, un savoir socioreprésenté. La distance entre savoir appris et savoir prescrit d’une part et le savoir savant d’autre part est telle qu’il est possible de parler d’une rupture épistémologique. Du point de vue méthodologique, la triangulation a montré l’intérêt et la spécificité de chaque méthode : le questionnaire classique ne permet pas d’appréhender la structuration d’une représentation mais offre de nombreuses informations nécessaires à la compréhension des données produites par les outils plus spécifiques comme les questionnaires d’évocations et de caractérisation. C’est la diversité des approches qui a permis de circonscrire le savoir de sens commun scolaire sur la préhistoire. Ce savoir est particulièrement homogène car il est en grande partie socialement partagé et historiquement transmis. Il est aussi le résultat de la position que chaque élève occupe dans le champ social et culturel : cette position contribue selon les variables en jeu à sa structuration. Il s’agit d’un savoir socioreprésenté plutôt qu’un savoir socioconstruit car au final l’influence de la pensée sociale est prépondérante face à l’absence d’un apport constructiviste qui aurait pu « élever » et « stabiliser » la pensée de sens commun vers une pensée plus rationnelle dite « pensée historienne scolaire ». La discipline savante de référence de la préhistoire, l’archéologie, nous semble être la ressource idéale dans laquelle puiser pour la mise en œuvre d’une pédagogie socioconstructiviste qui favoriserait le développement d’un mode de pensée rationnel. / Our work focuses on the representations of pupils in French school levels of “CE2”, “CM1” and “CM2” on prehistory and Cro-Magnon by articulating insights specific to the didactics of History (intermediate model of the appropriation of History) and social psychology (theory of social representations).We started from the principle that knowledge in History acquired at school was simplified to the extreme, full of common sense and sometimes in contradiction with the recent advances in prehistoric research. By doing so, school knowledge would be a socio-represented academic knowledge rather than a school knowledge socio-built essentially of educated RS. We made assumptions that pupils representations on prehistory was particularly unified under the influence of historical and socio-cultural social representations, despite the intervention of education. The programs and especially the manuals themselves filled with social representations would promote social thinking at the expense of a controlled historical thinking.We focused on methodological triangulation and conducted several types of analysis in order to confront them. After presenting the theoretical framework, we conducted a historical epistemological study on prehistory and Cro-Magnon with an analysis of historical representations from a bibliographic review, a synthesis of recent analyzes of representations of prehistory and a synthesis of current scientific data on prehistory. Analyses from the object of study identified the main epistemological obstacles and the underlying representations.We then proceeded to the analysis of empirical data that was collected in several ways: prioritized evocations questionnaires and characterization questionnaires. A more standard study using a classic questionnaire completed the device/scheme: this questionnaire distributed before, during and after the prehistory lesson for the CE2 level, enabled through a diachronic study to measure the influence of teaching on the pupils representations.The results were compared with those of the object of study and have made possible the identification of representations as well as the organizing principles of position statements, that is to say, their socio-cultural anchor.We showed that the meaning attributed to the object of social representation of Prehistory is the result of a confluence of factors where, in the end, teaching is only a marginal or a peripheral element. The major influence comes from the historical and sociocultural determinants. School knowledge of common sense developed after the teacher's intervention was close, in many ways, to the knowledge to be taught, because the latter is also, to a large extent, a socio-represented knowledge. The distance between learned knowledge and knowledge to be taught, on the one hand, and scientific knowledge on the other hand, is such that it is possible to speak of an epistemological rupture. From a methodological point of view, triangulation has showed the interest and the specificity of each method: the classic questionnaire does not allow to allow to understand the structuring of a representation but offers a lot of information necessary for the understanding of the data produced by more specific tools like evocations and characterization questionnaires. It is the diversity of approaches that helped identify the school knowledge of common sense on prehistory.Learning is not just the result of memory but an integration-reconstruction of knowledge elements. School knowledge of common sense on prehistory is particularly homogeneous because it is largely socially shared and historically transmitted. This knowledge is also the result of the position that each pupil occupies in the social and cultural fields: this position contributes according to variables involved (sex, socio-professional categories, level, geography, type of pedagogy, classroom / school) to its structuring. It is a socio-represented knowledge rather than a socio-b

This study uses the mindset of Bryan Eisenbergs four behavior types (Competitive, Spontaneous, Humanistic, and Methodical), to in- crease the conversion rate and the user experience while shopping online. Traditional retailers, i.e physical stores, are not a priority anymore and a challenge for an e-commerce site is to maintain a relationship to the customer online. The mindset of the four be- havior types is aimed to be used to target the universe of buyers. This mindset and theory will be applied to a CRO-design process, where the focus is to increase the conversion rate optimization of a website. An extensive literature study, an analysis phase, user tests and a questionnaire were used to decide whether this concept is worth using as a part of the process or not. The results indicate that the concept of the four behavior types can be used to target the audience. The thesis states that the concept could be used as a general foundation to further develop this mindset.

<p>DNA-recognizing proteins are involved in a multitude of important life-processes. Therefore, it is of great interest to understand the underlying mechanisms that set the rules for sequence specific protein–DNA interactions. Previous attempts aiming to resolve these interactions have been focused on naturally occurring systems. Due to the complexity of such systems, conclusions about structure–function relationship in protein–DNA interactions have been moderate. </p><p>To expand the knowledge of protein–DNA recognition, we have utilized<i> in vitro</i> evolution techniques. A phage display system was modified to express the DNA-binding, helix-turn-helix protein Cro from bacteriophage λ. A single-chain variant of Cro (scCro) was mutated in the amino acid residues important for sequence-specific DNA-binding. Three different phage-libraries were constructed. </p><p>Affinity selection towards a synthetic ORas12 DNA-ligand generated a consensus motif. Two clones containing the motif exhibited high specificity for ORas12 as compared to control ligands. The third library selection, based on the discovered motif, generated new protein variants with increased affinity for ORas-ligands. Competition experiments showed that Arg was important for high affinity, but the affinity was reduced in presence of Asp or Glu. By measuring <i>K</i>_D values of similar variant proteins, it was possible to correlate DNA-binding properties to the protein structure.</p><p>mRNA display of scCro was also conducted. The system retained the wild-type DNA-binding properties and allowed for functional selection of the mRNA–scCro fusion. Selected species was eluted and the gene encoding the scCro was recovered by PCR. </p><p>The two <i>in vitro</i> selection methods described in this thesis can be used to increase the knowledge of the structure–function relationship regarding protein–DNA recognition. Furthermore, we have also shown that new helix-turn-helix proteins exhibiting novel DNA-binding specificity can be constructed by phage display. The ability to construct proteins with altered DNA-specificity has various important applications in molecular biology and in gene therapy.</p>

DNA-recognizing proteins are involved in a multitude of important life-processes. Therefore, it is of great interest to understand the underlying mechanisms that set the rules for sequence specific protein–DNA interactions. Previous attempts aiming to resolve these interactions have been focused on naturally occurring systems. Due to the complexity of such systems, conclusions about structure–function relationship in protein–DNA interactions have been moderate. To expand the knowledge of protein–DNA recognition, we have utilized in vitro evolution techniques. A phage display system was modified to express the DNA-binding, helix-turn-helix protein Cro from bacteriophage λ. A single-chain variant of Cro (scCro) was mutated in the amino acid residues important for sequence-specific DNA-binding. Three different phage-libraries were constructed. Affinity selection towards a synthetic ORas12 DNA-ligand generated a consensus motif. Two clones containing the motif exhibited high specificity for ORas12 as compared to control ligands. The third library selection, based on the discovered motif, generated new protein variants with increased affinity for ORas-ligands. Competition experiments showed that Arg was important for high affinity, but the affinity was reduced in presence of Asp or Glu. By measuring KD values of similar variant proteins, it was possible to correlate DNA-binding properties to the protein structure. mRNA display of scCro was also conducted. The system retained the wild-type DNA-binding properties and allowed for functional selection of the mRNA–scCro fusion. Selected species was eluted and the gene encoding the scCro was recovered by PCR. The two in vitro selection methods described in this thesis can be used to increase the knowledge of the structure–function relationship regarding protein–DNA recognition. Furthermore, we have also shown that new helix-turn-helix proteins exhibiting novel DNA-binding specificity can be constructed by phage display. The ability to construct proteins with altered DNA-specificity has various important applications in molecular biology and in gene therapy.

The Swedish pharmaceutical industry is going through structural changes due to the increased cost of drug development. This has led pharmaceutical firms to outsource clinical trials to Clinical Research Organisations (CRO). This thesis explores the inter-firm relationships between pharmaceutical firms and CROs in the Stockholm area, with focus on the development of trust and commitment. The empirical material gathered from nine qualitative interviews with representatives from both parties suggests that trust and commitment are both important factors. Trust is based on competence and reputation whilst commitment is developed through communication. Commitment is highly valued by the companies that wished to develop long-term relationships. The results also point to challenges in the area mainly regarding patient recruitment that due to the high competition amongst the CROs can lead to opportunism when they are overly optimistic of what they can deliver. This is an issue that needs to be addressed as it affects the whole industry.

As the bio-pharmaceutical industries find itself moving forward in super high speed, the more strategic move it need to take to keep it survival in the biotechnology competition.The big pharm starts to release new drug exploring,investigation,production,and marketing and try to take the contract research method to run their business.Thus the Contract Research Organization (CRO) find its wave in the bio-pharmaceutical industries. The CRO in Taiwan is a young competitor.The CRO business is a knowledge-based incentive business.It takes medical human resources and stastics experts to make it run .It needs to qualify the national regulation while the clinical trial related regulations in Taiwan needs to qualify the international good clinical trial regulations.This study focuses on the competitive forces analysis of the CRO industries in Taiwan. The study will review the lately development of CRO companies in Taiwan.The study will review the company profile and analyze it in the Diamond model by Michael Porter. The government factor plays a significant role in the Clinical trial related business.The CRO will be more flexible and competitive under preper regulations.