Chinese hamster ovary (CHO) cells are frequently used for the commercial expression of recombinant therapeutic antibodies due to their ability to perform appropriate post-translational modifications and therefore generate an accurate rendition of natural products. As a consequence of their initial derivation by mutagenesis and the divergence into distinct cell lines, clonally-derived cell lines are phenotypically distinct. Molecular understanding of the features that determine the properties of a CHO clone is fundamental to the optimisation of cell environment and, potentially, engineering or selection of CHO clones with the “best” phenotype.The profile of the endoplasmic reticulum (ER) environment (with a specific complement of chaperones, co-chaperones, and sensors) is important for cell growth and maximum recombinant protein secretion. I have addressed how the modulation of two components in the ER, XBP1(s) and ERO1L α, influence CHO cell function. XBP1(s) is generated by a novel mRNA splicing mechanism in response to ER stress and is the key regulator factor for the development of professional secretory cells. ERO1L α plays a critical role in setting the redox state of foldases (such as PDI) and is also known to be induced by ER stress. In this study, CHO S cells were doubly transfected with human XBP1(s) and human ERO1L α constructs to generate a series of CHO cell lines that overexpressed each gene. The engineered cell lines exhibited a series of improvements in terms of desirable phenotypes compared to the non-engineered CHO S cell line. These improvements included up-regulation of chaperone expression, alteration in growth profile and associated glucose consumption and lactate production, increased antibody titres, and improved recovery from an oxidative stress. My interpretation is that engineering cells to over-express XBP1(s) and ERO1L α generated a more favourable phenotype for cells to handle the stresses that result from protein transit in the ER. Whether this is a direct effect of XBP1(s) and ERO1L α or due to a secondary consequence of their over-expression on the ER chaperone complement remains unclear. However, this study identified important combinations of regulatory factors that influence ER function and, consequently, the ability to define improved CHO cell phenotypes for expression of different types of protein products.

<b><u>Purpose</u></b>: Some randomized trials are reported widely, while others remain unpublished. It is essential to systematic reviewers and meta-analysts that factors leading to publication bias in the form of delayed or non-publication of an eligible study are identified. This thesis is an attempt to do this. <br></br><br></br> <b><u>Data</u></b>: The set of randomized trials identified by the Childhood Acute Lymphoblastic Leukaemia (ALL) Collaborative Group was used. This consists of 149 trials comprising 243 randomized comparisons (randomizations), starting prior to 1 January 1988, reported in 257 articles, published prior to 1 January 2000. Each mention of a randomization in an article (irrespective of whether results are given) generates a publication record, of which there are 610. <br></br><br></br> <b><u>Methods</u></b>: The main focus is on identifying which trial characteristics lead to a delay in publication of a randomization. Time to the first mention of a randomization in an article (irrespective of whether any results are given) and to the first reporting of its results are both modelled using ordinary linear regression (the independence model). However, when these analyses are extended to include all mentions and all reportings of results respectively, non-independence necessitates the use of techniques for dealing with repeated measures. In such cases the independence model is the starting point, the residuals from which are used to form the covariance matrix, which in turn is used to suggest plausible correlation structures for repeated measures models. Generalised estimating equation (GEE) analysis is used to select an appropriate correlation structure, and a linear mixed effects model serves to confirm this. The conclusions are then discussed in the context of other studies identified. Finally logistic regression is used to identify trial characteristics associated with a randomization remaining unpublished, and Poisson and negative binomial models to identify those affecting frequency of reporting. <br></br><br></br> <b><u>Results</u></b>: Evidence was found of ‘pipeline bias’ in the reporting of first results since, although direction of effect was not found to be significant, highly statistically significant results are published faster than others. However this is not so for first mentions. Negative results (i.e. those in favour of the standard/control) arm were submitted for first publication faster than all others, although this did not effect time to publication. In addition, geographic location is an important predictor of whether a randomization is ever mentioned in an article, frequency of mentions and of time to first publication and results from single-centre trials are published more frequently than those with multi-centre participation. <br></br><br></br> <b><u>Conclusions</u></b>: Although ‘pipeline bias’ was identified in the analysis of time first reporting of results, it was not present in the analysis of time to first mention, and so not a problem for those wishing only to identify randomized trials for inclusion in meta-analyses. The importance of geographic location suggests that the practice of contacting known trialists is worthwhile in addition to the computerised literature searches and should be continued. <br></br><br></br>

Les cellules souches embryonnaires (ESCs) sont issues de la masse cellulaire interne (ICM) de blastocystes préimplantatoires. Elles sont pluripotentes c'est-à-dire capables de se différencier dans les trois lignages embryonnaires (ectoderme, mésoderme et endoderme) et de s'autorenouveller, c'est-à-dire de se multiplier indéfiniment en culture. Chez la souris, ces cellules (mESCs) sont à la base des techniques de transgénèse permettant des modifications génétiques ciblées. Chez l'Homme ces cellules (hESCs) représentent un grand espoir en médecine régénérative pour traiter des maladies dégénératives comme les maladies de Parkinson ou de Huntington. Le modèle le plus pertinent de l'espèce humaine est le singe. Cependant l'expérimentation sur cette espèce est soumise à une réglementation très stricte. C'est pourquoi il est nécessaire de développer des modèles alternatifs. C'est dans ce cadre que s'inscrit le lapin, qui est phylogénétiquement plus proche de l'Homme que ne l'est la souris. Mon projet de thèse a eu pour but d'étudier la pluripotence dans les ESCs de lapin (rESCs), afin de pouvoir les utiliser en transgénèse et produire des animaux transgéniques, modèles de maladies humaines. La première partie de ces analyses est regroupée au sein de l'article que notre laboratoire a publié en 2013 dans Biology Open (Osteil et al. 2013). D'autres analyses ont abouti à la dérivation de nouvelles lignées stabilisées dans un état plus proche de celui des cellules de l'ICM. L'ensemble des résultats a permis d'établir des bases solides pour la compréhension de la pluripotence et pour la dérivation d'ESCs dites naïves chez un autre mammifère que la souris / Embryonic stem cells (ESCs) result from cultures of inner cell masses (ICMs) isolated at preimplantation blastocyst stage. ESCs are defined by their self-renewal capacity, characterized by robust proliferation while maintaining plutipotent potential, the ability to give rise to cells from all three germ layers mesoderm, endoderm and ectoderm. Mouse ESCs (mESCs) allow the production of transgenic models by site-specific mutagenesis. Human ESCs (hESCs) represent major hope for regenerative medicine in order to treat degenerative diseases like Parkinson or Huntington. The more relevant model of Human is monkey. However, working on this specie is subjected to extremely strict regulation. Consequently it is very important to develop alternative animal models. Rabbit appears to be a very good candidate, because he is phylogenetically closer to Human than the mouse. My thesis project aimed to study the pluripotency mechanism of rabbit ESCs (rESCs), in order to use these cells for the production of transgenic animal models for human diseases. First part of theses analyses is synthesized in a publication into Biology Open in 2013 (Osteil et al. 2013). Other analyses produced new rESCs lines stabilized in a closer state compared to ICM state. All these results led to obtain solid knowledge on pluripotency and derivation on so-called naïve ESCs in a non-rodent specie