Methods of genetic diversity creation and functional display for directed evolution experiments

Protein engineering aims to improve the properties of enzymes and affinity reagents by genetic changes. Typical engineered properties are affinity, specificity, stability, expression, and solubility. Because proteins are complex biomolecules, the effects of specific genetic changes are seldom predictable. Consequently, a popular strategy in protein engineering is to create a library of genetic variants of the target molecule, and render the population in a selection process to sort the variants by the desired property. This technique, called directed evolution, is a central tool for trimming protein-based products used in a wide range of applications from laundry detergents to anti-cancer drugs. New methods are continuously needed to generate larger gene repertoires and compatible selection platforms to shorten the development timeline for new biochemicals. In the first study of this thesis, primer extension mutagenesis was revisited to establish higher quality gene variant libraries in Escherichia coli cells. In the second study, recombination was explored as a method to expand the number of screenable enzyme variants. A selection platform was developed to improve antigen binding fragment (Fab) display on filamentous phages in the third article and, in the fourth study, novel design concepts were tested by two differentially randomized recombinant antibody libraries. Finally, in the last study, the performance of the same antibody repertoire was compared in phage display selections as a genetic fusion to different phage capsid proteins and in different antibody formats, Fab vs. single chain variable fragment (ScFv), in order to find out the most suitable display platform for the library at hand. As a result of the studies, a novel gene library construction method, termed selective rolling circle amplification (sRCA), was developed. The method increases mutagenesis frequency close to 100% in the final library and the number of transformants over 100-fold compared to traditional primer extension mutagenesis. In the second study, Cre/loxP recombination was found to be an appropriate tool to resolve the DNA concatemer resulting from error-prone RCA (epRCA) mutagenesis into monomeric circular DNA units for higher efficiency transformation into E. coli. Library selections against antigens of various size in the fourth study demonstrated that diversity placed closer to the antigen binding site of antibodies supports generation of antibodies against haptens and peptides, whereas diversity at more peripheral locations is better suited for targeting proteins. The conclusion from a comparison of the display formats was that truncated capsid protein three (p3Δ) of filamentous phage was superior to the full-length p3 and protein nine (p9) in obtaining a high number of uniquely specific clones. Especially for digoxigenin, a difficult hapten target, the antibody repertoire as ScFv-p3Δ provided the clones with the highest affinity for binding. This thesis on the construction, design, and selection of gene variant libraries contributes to the practical know-how in directed evolution and contains useful information for scientists in the field to support their undertakings.

Association between Information and Communication Technology Usage and the Quality of Sleep among School-Aged Children during a School Week

ilm OKM 2013

Backpacker 2.0 - Contemporary backpackers and their use of social media as an information and communication platform

Long-term independent budget travel to countries far away has become increasingly common over the last few decades, and backpacking has now entered the tourism mainstream. Nowadays, backpackers are a very important segment of the global travel market. Backpacking is a type of tourism that involves a lot of information search activities. The Internet has become a major source of information as well as a platform for tourism business transactions. It allows travelers to gain information very effortlessly and to learn about tourist destinations and products directly from other travelers in the form of electronic word-of-mouth (eWOM). Social media has penetrated and changed the backpacker market, as now modern travelers can stay connected to people at home, read online recommendations, and organize and book their trips very independently. In order to create a wider understanding on modern-day backpackers and their information search and share behavior in the Web 2.0 era, this thesis examined contemporary backpackers and their use of social media as an information and communication platform. In order to achieve this goal, three sub-objectives were identified: 1. to describe contemporary backpacker tourism 2. to examine contemporary backpackers’ travel information search and share behavior 3. to explore the impacts of new information and communications technologies and Web 2.0 on backpacker tourism The empirical data was gathered with an online survey, thus the method of analysis was mainly quantitative, and a qualitative method was used for a brief analysis of open questions. The research included both descriptive and analytical approaches, as the goal was to describe modern-day backpackers, and to examine possible interdependencies between information search and share behavior and background variables. The interdependencies were tested for statistical significance with the help of five research hypotheses. The results suggested that backpackers no longer fall under the original backpacker definitions described some decades ago. Now, they are mainly short-term travelers, whose trips resemble more those of mainstream tourists. They use communication technologies very actively, and particularly social media. Traditional information sources, mainly guide books and recommendations from friends, are of great importance to them but also eWOM sources are widely used in travel decision making. The use of each source varies according to the stage of the trip. All in all, Web 2.0 and new ICTs have transformed the backpacker tourism industry in many ways. Although the experience has become less authentic in some travelers’ eyes, the backpacker culture is still recognizable.

Task Force on Librarians’ Competencies in Support of E-Research and Scholarly Communication

Presentation at Open Repositories 2014, Helsinki, Finland, June 9-13, 2014

The Purdue University Research Repository (PURR): An institutional data management service with a virtual research environment, data publication, and archiving

Poster at Open Repositories 2014, Helsinki, Finland, June 9-13, 2014

The Human Communication Science Virtual Lab (HCS vLab): A repository microclimate in a rapidly evolving research-ecosystem

Presentation at Open Repositories 2014, Helsinki, Finland, June 9-13, 2014

Scheduling dynamic dataflow graphs with model checking

With the shift towards many-core computer architectures, dataflow programming has been proposed as one potential solution for producing software that scales to a varying number of processor cores. Programming for parallel architectures is considered difficult as the current popular programming languages are inherently sequential and introducing parallelism is typically up to the programmer. Dataflow, however, is inherently parallel, describing an application as a directed graph, where nodes represent calculations and edges represent a data dependency in form of a queue. These queues are the only allowed communication between the nodes, making the dependencies between the nodes explicit and thereby also the parallelism. Once a node have the su cient inputs available, the node can, independently of any other node, perform calculations, consume inputs, and produce outputs. Data ow models have existed for several decades and have become popular for describing signal processing applications as the graph representation is a very natural representation within this eld. Digital lters are typically described with boxes and arrows also in textbooks. Data ow is also becoming more interesting in other domains, and in principle, any application working on an information stream ts the dataflow paradigm. Such applications are, among others, network protocols, cryptography, and multimedia applications. As an example, the MPEG group standardized a dataflow language called RVC-CAL to be use within reconfigurable video coding. Describing a video coder as a data ow network instead of with conventional programming languages, makes the coder more readable as it describes how the video dataflows through the different coding tools. While dataflow provides an intuitive representation for many applications, it also introduces some new problems that need to be solved in order for data ow to be more widely used. The explicit parallelism of a dataflow program is descriptive and enables an improved utilization of available processing units, however, the independent nodes also implies that some kind of scheduling is required. The need for efficient scheduling becomes even more evident when the number of nodes is larger than the number of processing units and several nodes are running concurrently on one processor core. There exist several data ow models of computation, with different trade-offs between expressiveness and analyzability. These vary from rather restricted but statically schedulable, with minimal scheduling overhead, to dynamic where each ring requires a ring rule to evaluated. The model used in this work, namely RVC-CAL, is a very expressive language, and in the general case it requires dynamic scheduling, however, the strong encapsulation of dataflow nodes enables analysis and the scheduling overhead can be reduced by using quasi-static, or piecewise static, scheduling techniques. The scheduling problem is concerned with nding the few scheduling decisions that must be run-time, while most decisions are pre-calculated. The result is then an, as small as possible, set of static schedules that are dynamically scheduled. To identify these dynamic decisions and to find the concrete schedules, this thesis shows how quasi-static scheduling can be represented as a model checking problem. This involves identifying the relevant information to generate a minimal but complete model to be used for model checking. The model must describe everything that may affect scheduling of the application while omitting everything else in order to avoid state space explosion. This kind of simplification is necessary to make the state space analysis feasible. For the model checker to nd the actual schedules, a set of scheduling strategies are de ned which are able to produce quasi-static schedulers for a wide range of applications. The results of this work show that actor composition with quasi-static scheduling can be used to transform data ow programs to t many different computer architecture with different type and number of cores. This in turn, enables dataflow to provide a more platform independent representation as one application can be fitted to a specific processor architecture without changing the actual program representation. Instead, the program representation is in the context of design space exploration optimized by the development tools to fit the target platform. This work focuses on representing the dataflow scheduling problem as a model checking problem and is implemented as part of a compiler infrastructure. The thesis also presents experimental results as evidence of the usefulness of the approach.

Target audience and message alignment for integrated marketing communication of luxury products: entering the Chinese market

The purpose of this study is to identify opportunities to match marketing communication message strategies with the target audience characteristics in the Chinese luxury market entry context. Therefore, consumer behaviour and psychographic marketing segmentation fields are being reviewed in a holistic view in order to identify the similarities and connection points. Through the analysis of the messages in advertisements placed in a certain luxury and fine living magazine, message creation strategies are being anticipated.

The effect of celebrity CEO’s crisis communication to crisis outcome

The purpose of this this study is to examine the effects that a celebrity CEO’s crisis communication has on the crisis outcome and further on the quick decision response, resource mobilization and information flow during a crisis. The research was conducted using a multiple embedded case method, with four cases that represent different kinds of crisis that have all involved a celebrity CEO. The cases have been collected from various secondary data sources. The results of the study indicate that in crisis situations the celebrity CEO is expected to take responsibility of the crisis communication. The CEO needs to understand how and what to communicate in these situations and they need to make quick decisions even with limited information available. Crisis outcome is highly dependent on the golden hour crisis communication and there the performance of the celebrity CEO is crucial.

Suorituskyvyn johtaminen ja IT-strategian suuntaaminen; Case: ICT-alan yritys

Yrityksen tietohallintoon kohdistuu tänä päivänä paljon vaatimuksia. Vaatimukset kohdistuvat tietohallinnon merkitykseen liiketoiminnan tukijana ja suorituskyvyn edistäjänä. ICT-toimialan rooli palveluiden tuottajana ja Suomen taloudellisen kas-vun tukijana ovat merkittävät. Tämän diplomityön tavoite on selvittää mikä on tietohallinnon rooli ja merkitys osana yrityksen suorituskykyä ja miten sitä tulisi kehittää. Tutkimuksen teoreettinen viitekehys koostuu yleisistä suorituskyvyn johtamisen malleista, suorituskyvyn joh-tamisen malleista tietohallinnossa sekä IT-strategian suuntaamisen malleista ja suuntaamisen vaikutuksesta yrityksen suorituskykyyn. Tutkimuskysymys on: miten strategian suuntaamista tulisi kehittää jotta tietohallinto pystyisi edistämään yrityk-sen suorituskykyä? Tutkimusstrategia on case-tutkimus ja empiirinen aineisto on hankittu teemahaastatteluilla. Case-tutkimuksen tulosten mukaan strategian suuntaamisen arvioinnin mallin valit-seminen on tärkeää. Strategian suuntaamisen kaikki näkökulmat tulee ottaa huomi-oon. Esimerkiksi osaaminen, kommunikaatio, kumppanuudet ja ihmisten käyttäy-tyminen saattavat jäädä ns. kovien näkökulmien varjoon. Strategian suuntaamisessa tulee kiinnittää huomiota suuntaamiseen kaikilla organisaatiotasoilla. Toiminnalli-sen tason merkitys tulee myös huomioida. Strategian suuntaamista kehitettäessä tulee arvioinnissa ottaa huomioon eri liiketoimintayksiköiden arvioinnit, koska ne saattavat poiketa vahvasti toisistaan ja siten vääristää kehittämiskohteiden valitse-mista.

Comparing adult-youth and adult-adult online sexual solicitation: Manipulative behaviors, situational factors, and outcomes

Online sexual solicitation (solicitation) of youth has received widespread media and research attention during the last decade. The prevalence rates of youth who have experienced solicitation or solicitation attempts vary between studies depending on the methodology used (e.g., whether youth or adults are the target study group). In studies focusing on youth victims, the prevalence of solicitation attempts made by adults during the past year is typically reported to be between 5 and 9%. Adults who solicit youth online have been found to use deception and other manipulative behaviors to gain access to sexual activities with youth. However, previous studies have lacked a control group of adults who solicit other adults online. Without this comparison, one could argue that deceiving others online about one’s identity, and engaging in manipulative behaviors, is an inherent part of most online sexual interactions with strangers. Additionally, little is known about the associations between manipulative behaviors and the solicitation outcomes. In research concerning offline sexual behaviors, it has been noted that situational factors, such as sexual arousal, may alter both sexual interest and behavior. The effects of situational factors on online sexual behaviors have been less extensively studied (especially so with a quantitative approach); no studies have to date focused on adults’ solicitation of youth. Investigating the role of a lowered sexual age preference and the role of situational factors in the soliciting adults could be an important step in order to receive deeper knowledge of the role of traits and states in the context of solicitation. Additionally, there is a lack of knowledge of the effect of the age of the youth. Although previous studies on solicitation has found that older youth, compared with younger youth and children, are more often solicited, the possible reasons for this have not been investigated. Are adults who solicit youth affected by legal deterrence (through the legal age of consent), is it because older youth are more available online, or are the adults’ age preferences merely a product of a normally distributed age preference in the population? The purpose of the present thesis was fivefold: 1) to obtain an estimate of the frequency of adults’ solicitation of youth as self-reported and observed in actual behavior; 2) to explore whether the legal age of consent (LAC) affects solicitation frequency, or whether a normally distributed sexual age preference more accurately describe the proportion of solicited youth of different ages; 3) to investigate the associations of both traits (e.g., lower sexual age preference) and states (immediate situational factors, such as alcohol intoxication), and the solicitation target; 4) to explore whether adults who solicit youth and adults who solicit adults are equally deceitful and manipulative online, and whether the different solicitation outcomes are as common in both groups; and 5) to investigate whether the deceitful and manipulative behaviors engaged in had different associations with the solicitation outcomes depending on the age of the solicited. In the survey study, a convenience sample of 1393 adult participants (aged 18 years or older) self-reported any online communication with strangers during the past year. Of these, 56% (776 respondents) reported that they had solicited or attempted to solicit at least one stranger. Of the respondents, 453 (58.4%) were men, and 323 (41.6%) were women. Participants with only adult contacts (18 years or older) constituted the majority (640 respondents). In contrast, 136 individuals reported a youth contact (a 13 year old or younger, or a 14 to 17-year old). Approximately half of the participants were men in the adult contact group, while 75% of the participants were men in the youth contact group. Approximately 60% of the participants with youth contacts were recruited from two websites associated with a pedophilic sexual interest. In an online quasi-experimental study, with researchers impersonating youth of different ages (10–18 year olds) in chat rooms, 251 online conversations with chat room visitors made up the entire sample. All chat room visitors alleged to be men. The self-reported frequency of having solicited youth (0–17-year olds) during the past year was approximately 10% in our sample of adults who reported communicating with any strangers online. When we observed this behavior in chat rooms, we found that approximately 30% of the chat room visitors who believed they interacted with a 10 to 14 year old attempted to solicit the youth. We found that solicitation attempts increased equally much when increasing the age of the impersonated youth from 14 to 16, as from 16 to 18. Thus, we concluded that a normally distributed age preference in the population was a more plausible explanation to the effect of the age of the solicited, rather than the LAC (here; 15 and 16). If the chat room visitors would have been deterred only by the LAC, we would have expected that the change in amount of solicitation attempts from an illegal age group to a legal age group would have been significantly stronger than changes between age groups within illegal-illegal and legal-legal groups. Our subsample of survey participants from the pedophilia-related websites expectedly reported that they had solicited youth more often in comparison to the sample gathered through general (i.e., not associated with any particular sexual preference) websites. We also found that participants with a youth contact reported higher levels of sexual arousal and shame before the sexual interaction with their online contact, compared with participants with an adult contact. Additionally, the participants with youth contacts who reported consumption of child- and adolescent pornography also reported being more sexually aroused before the interaction, compared to the participants with youth contacts who did not report consumption of these kinds of pornography. We also found clear indications that the online sexual interaction had an alleviatory effect on reported levels of sadness, boredom and stress, independent of the age of the contact. Generally, the participants with youth and adult contacts reported deceiving their contacts as often and suggesting keeping the communication a secret from someone as often. Participants with a youth contact, however, reported using more persuasion techniques for online sexual purposes or for the purpose of an offline meeting, compared to those with an adult contact. In the chat rooms, we found that more indirect ways of future sexual communication (e.g., continuing chatting) was suggested by the chat room visitors that were under the assumption of interacting with youth aged 10 to 14, compared with more direct means (e.g., meeting offline). Survey participants with youth contacts who had used deception, suggested keeping the interactions a secret, and/or persuaded their contact by appealing to the contacts feelings of love and attachment for the participant had also more often engaged in cybersex with the contact. No other manipulative behaviors were associated with the other investigated solicitation outcomes (receiving a sexual picture, meeting offline, and engaging in sexual contact offline) within this group of participants. However, using deception, suggesting secrecy and using persuasion was also positively associated with certain solicitation outcomes within participants with an adult contact. In summary, adults’ solicitation of youth is much more frequent when observed in chat rooms than self-reported. Additionally, an underlying lowered sexual age preference seems to be a motivating factor on a group level in adults who solicit youth. We concluded that directed prevention efforts should be made on pedophiliarelated websites. Additionally, the role of situational factors, especially sexual arousal in persons with a pedo- or hebephilic sexual interest should be investigated further in the context of online sexual solicitation.