Discovery and evolution of novel Cre-type tyrosine site-specific recombinases for advanced genome engineering

Tyrosine site-specific recombinases (Y-SSRs) are DNA editing enzymes that play a valuable role for the manipulation of genomes, due to their precision and versatility. They have been widely used in biotechnology and molecular biology for various applications, and are slowly finding their spot in gene therapy in recent years. However, the limited number of available Y-SSR systems and their often narrow target specificity have hindered the full potential of these enzymes for advanced genome engineering. In this PhD thesis, I conducted a comprehensive investigation of novel Y-SSRs and their potential for advancing genome engineering. This PhD thesis aims to address the current limitations in the genetic toolbox by identifying and characterizing novel Cre-type recombinases and demonstrating their impact on the directed evolution of designer recombinases for precise genome surgery. To achieve these aims, I developed in a collaboration a comprehensive prediction pipeline, combining a rational bioinformatical approach with knowledge of the biological functions of recombinases, to enable high success rate and high-throughput identification of novel tyrosine site-specific recombinase (Y-SSR) systems. Eight putative candidates were molecularly characterized in-depth to ensure their successful integration into future genome engineering applications. I assessed their activity in prokaryotes (E. coli) and eukaryotes (human cell lines), and determined their specificity in the sequence space of all known Cre- type target sites. The potential cytotoxicity associated with cryptic genomic recombination sites was also explored in the context of recombinase applicability. This approach allowed the identification of novel Y-SSRs with distinct target sites, enabling simultaneous use of multiple Y-SSR systems, and provided knowledge that will facilitate the assignment of novel and known recombinases to specific uses or organisms, ensuring their safe and effective implementation. The introduction of these novel Y-SSRs into the genome engineering toolbox opens up new possibilities for precise genome manipulation in various applications. The broader targetability offered by these enzymes could accelerate the development of novel gene therapies, as well as advance the understanding of gene function and regulation. Moreover, these recombinases could be used to design custom genetic circuits for synthetic biology, allowing researchers to create more complex and sophisticated cellular systems. Finally, I introduced the novel Y-SSRs into efforts aimed at developing designer recombinases for precise genome surgery, demonstrating their impact on accelerating the directed evolution process. Therapeutically relevant recombinases with altered DNA specificity have been developed for excision or inversion of specific DNA sequences. However, the potential for evolving recombinases capable of integrating large DNA cargos into naturally occurring lox-like sites in the human genome remained untapped so far. Thus, I embarked on evolving the Vika recombinase to mediate the integration of DNA cargo into a native human sequence. I discovered that Vika could integrate DNA into the voxH9 site in the human genome, and then, I enhanced the process through directed evolution. The evolved variants of Vika displayed a marked improvement in integration efficiency in bacterial systems. However, the translation of these results into mammalian systems has not yet been entirely successful. Despite this, the study laid the groundwork for future research to optimize the efficiency and applicability of Y-SSRs for genomic integration. In summary, this thesis made significant strides in the identification, characterization, and development of novel Y-SSRs for advanced genome engineering. The comprehensive prediction pipeline, combined with in-depth molecular characterization, has expanded the genetic toolbox to meet the growing demand for better genome editing tools. By exploring efficiency, cross-specificity, and potential cytotoxicity, this research lays the foundation for the safe and effective application of novel Y-SSRs in various therapeutic settings. Furthermore, by demonstrating the potential of these recombinases to improve efforts in creating designer recombinases through directed evolution, this research has opened new avenues for precise genome surgery. The successful development and implementation of these novel recombinases have the potential to revolutionize gene therapy, synthetic biology, and our understanding of gene function and regulation

Unconscious auditory information-processing during general anaesthesia

This dissertation aims to examine the possibility of cognitive processing and memory storage in anaesthesia. It consists of four parts. The first section provides a brief outline of unconscious mental processes in psychological research. Next, a review of the experimental studies of unconscious perception duting anaesthesia is given. The third part contains four empirical investigations of stimulus registration in anaesthetized patients. Finally, the research findings are evaluated, and implications for clinical practice in anaesthesiology and suggestions for future research are presented

Learning and improvig languages for the milennial generation

The idea of my Bachelor thesis was born while I was in Erasmus in Madrid, Spain. In this international environment, I discovered that it was most complicated than I thought to learn a new language and to meet locals. With a friend, we started wondering what could be an adapted tool to help us learning and meeting new people for our generation, the so-called “Millennials”. I had the idea to create an app, that would allow local people and exchange student to meet and speak. I would call it “Meak”. Millennials is the greatest generation in the US and one of the largest in history. This generation has really specific characteristics that have to be understood in order to create products and services that are adapted to its needs and wants. We analyzed those characteristics and could highlight the following points about Millennials: • Multicultural • Digital natives • Internationals • Spending habits and skepticism In this context, an app could match with Millennials behaviors and needs. To ensure the app market is propitious to the launch of a startup, we looked closer at the business environment. It appeared to be crowded but mature. With efficient communication and marketing, it is an interesting market. Then, we designed the features that our app should contains by running surveys and interviews. We adapted our app to our findings. Once the app was designed, we had to ensure the business model would be viable and generate enough revenue. To do so, we used the business model canvas and realized the application is profitable. Finally, we made a prototype that has been shown to potential customers. We gathered feedback and explained which changed had to be done to match with their expectations

Effect of self-rated health on cognitive performance in community dwelling elderly

The objective of this study was to examine the effect of self-reported health on. cognitive function in community dwelling elderly (N = 4,528). Research participants were divided into four groups with regard to self-rated health. Statistically controlling for the effects of depression, age, and education, participants with poor self-reported health harl lower scores on the Mini-Mental Status Examination than those who believed themselves to be in good health. Our results show that cognitive performance in older adults is influenced by health factors.</p