Study of DNA repair and recombination mechanisms in Chinese hamster ovary cells

The CRISPR nuclease systems greatly facilitate targeted genome modifications in mammalian cells. The outcome of genome editing depends on the involved DNA double strand break (DSB) repair pathways. While the classical non-homologous end-joining and the poorly defined alternative end-joining (alt-EJ) DSB repair pathways can cause imprecise repair and thus gene inactivations, the homologous recombination (HR) pathway often introduces precise modifications. Although CRISPR is highly efficient at inactivating single genes, it is inefficient at introducing precise genome modifications. Moreover, its efficiency at inactivating multi-locus DNA sequences such as highly repetitive endogenous viral elements also remains limited. This thesis addressed these limitations by better characterizing DSB repair pathways in Chinese hamster ovary (CHO) cells - the most widely used production cell host for therapeutic proteins. In this thesis, I first aimed at identifying rate-limiting factors to improve HR-mediated genome editing. Second, I strove for studying approaches to inactivate repetitive endogenous retroviruses (ERV) presumably releasing viral particles into the CHO supernatant. To identify factors limiting HR, we established two chromosomal CHO assays that measure HR activity based on the correction of a GFP loss-of-function mutation. By using knockdown and overexpression studies, we found that efficient HR-mediated genome editing depended on certain alt-EJ activities. Furthermore, we observed that alt-EJ contribution to HR correlates with the nuclease type and the location of the DSB site relative to the GFP mutation. These observations suggest that alt-EJ and HR repair pathways tightly interact and challenges the common perception of alt-EJ opposing HR. Finally, among the tested repair factors, high Mre11 nuclease and Pari anti-recombinase as well as low Rad51 recombinase levels were the most rate-limiting factors for HR in CHO cells. Counteracting these bottlenecks improved HR efficiency by 75%. To inactivate repetitive ERVs, we transiently expressed a CRISPR-Cas9 nuclease that targets the gag gene of a specific transcriptionally active ERV group. Clones bearing a loss-of-function mutation in one particular ERV locus and corresponding mRNA produced considerably fewer particles loaded with viral RNA genomes. These findings indicated that a single ERV locus is responsible for the release of most, if not all, viral particles from CHO cells. Notably, ERV mutagenesis did not compromise cell growth, cell size or therapeutic protein production. In sum, this work provided novel strategies to improve HR-mediated genome editing and to inhibit viral particle release from CHO cells

Kinetic evidence for two different productive substrate binding sites in subtilisins

Public data made available to citizens must meet certain characteristics to guarantee it can be easily reused to generate new information or services and, ultimately, to increase the transparency of public bodies. This article examines the recognition of these characteristics in the Spanish legislation and analyses how this is reflected in the open data websites set up by Spanish public bodies in recent years through examining the legal notices on these open data websites provided by Spanish public entities at both the national and regional levels. Lastly, we make a range of proposals for guaranteeing the adequate reuse of this data.Los datos públicos que se ponen a disposición de los ciudadanos deben cumplir determinadas características que persiguen garantizar que se puedan reutilizar fácilmente para generar nueva información o servicios y, en última instancia, para incrementar la transparencia de las administraciones públicas. En este artículo se examina el reconocimiento de estas características en la legislación española y se analiza su reflejo en los portales de datos abiertos impulsados por las administraciones públicas españolas en los últimos años, a través de la lectura de los avisos legales que difunden los portales de datos abiertos de la Administración General del Estado y de las comunidades autónomas. Finalmente, se realizan diferentes propuestas para garantizar la adecuada reutilización de los datos

Together | Apart: Printmaking and the Space Between

In this practice-led creative research, being both artist and identical twin, I examine sameness and difference in the relationships between two people and between multiple objects/creative works. Through the inherent ability of printmaking processes to produce multiples and the attendant installation opportunities created by punctuated space, I unfold and re-tell a doubled and ambiguous understanding of being in the world. Martin Heidegger’s notion of Being-in-the-world (Dasein) underpins and situates my understanding of being in the world, thrown into a world as an individual, and my existence as a double: being in the world with others. I relate his thinking through Barbara Bolt’s interpretation of Heidegger’s Dasein to the artist’s making to seek new ways of understanding Dasein / being-there. The work I make is a metaphor for investigating how I understand the world around me. To do this, I discuss tacit methodology and draw together the ‘what’, ‘how’ and ‘why’, which forms and informs a praxical knowledge, a knowledge that comes from doing and its reflective dimension. By investigating practices by both historical and contemporary artists who employ the diptych as a device to present works as an interconnected pair, I position my forms of the diptych to create relationships of closeness and separation, together | apart. The space in between two people or artworks—a gap—becomes instrumental in separating while simultaneously relating to and evoking continuation and connection. Imagery derived from and alluding to the body, in a gap between figuration and abstraction, is developed in an iterative, open-ended series of prints. Academic discussions about the nature of studio research are applied and interpreted through creative practice, imparting and enabling both an informed, personally situated perspective and an appraisal of the engagement of other artists, such as Roni Horn, Lesley Duxbury, and Paul Uhlmann, whose practices of ambiguity, in-between spaces and gaps are embedded in the wider field of visual arts practices. This research contributes to a broader field of discussion of understandings of being in the world, of spaces in-between and impermanence as related to unique printmaking practices

Geringe Samenbank von beweidbaren Arten für die Etablierung von Waldweiden im Schweizer Mittelland

Kipfer T. and Bosshard A. 2007. Low seed bank of herb species suitable for grazing hampers the establishment of wood pastures in the Swiss lowlands. Bot. Helv. 117: 159 - 167. Controlled forest grazing is expected to yield benefits for biodiversity conservation, landscape quality, and in some cases also for land use economy. In the Swiss lowlands, first attempts are being made to reintroduce forest grazing in productive beech forests, but methodic experience is still limited. One main issue concerns the development of the vegetation after forest stands have been thinned to improve light conditions: Will grassland vegetation establish spontaneously? The present study analyses the composition of the soil seed bank of four beech forest stands. The seed bank density ranged from 1'244 to 28'651 seeds m−2. Seed banks mainly consisted of forest and ruderal species; most abundant were Juncus effusus, Carex sylvatica, Rubus spp. and Clematis vitalba. Seeds of grassland plants were restricted to a few species, and their abundance in the seed bank decreased rapidly with increasing distance from the forest edge. These results reveal that there is little potential for grasslands to develop spontaneously from the seed bank. The introduction of grassland species of local origin using the green hay method is therefore recommended to prevent soil degradation during the first years of grazing, to fulfil minimal biodiversity requirements and to lower the risk of an establishment of neophytes and other problematic plant specie