Proceedings of the Schistosome Genome Project

"The host-parasite relationship" is a vast and diverse research field which, despite huge human and financial input over many years, remains largely shrouded in mystery. Clearly, the adaptation of parasites to their different host species, and to the different environmental stresses that they represent, depends on interactions with, and responses to, various molecules of host and/or parasite origin. The schistosome genome project is a primary strategy to reach the goal; this systematic research project has successfully developed novel technologies for qualitative and quantitative characterization of schistosome genes and genome organization by extensive international collaboration between top quality laboratories. Schistosomes are a family of parasitic blood flukes (Phylum Platyhelminthes), which have seven pairs of autosomal chromosomes and one pair of sex chromosomes (ZZ for a male worm and ZW for a female), of a haploid genome size of 2.7x108 base pairs (Simpson et al. 1982). Schistosomes are ideal model organisms for the development of genome mapping strategies since they have a small genome size comparable to that of well-characterized model organisms such as Caenorhabditis elegans (100 Mb) and Drosophila (165 Mb), and contain functional genes with a high level of homology to the host mammalian genes. Here we summarize the current progress in the schistosome genome project, the information of 3,047 transcribed genes (Expressed Sequence Tags; EST), complete sets of cDNA and genomic DNA libraries (including YAC and cosmid libraries) with a mapping technique to the well defined schistosome chromosomes. The schistosome genome project will further identify and characterize the key molecules that are responsible for host-parasite adaptation, i.e., successful growth, development, maturation and reproduction of the parasite within its host in the near future

Immune responses of IL-5 transgenic mice to parasites and aeroallergens

Eosinophils have long been thought to be effectors of immunity to helminths but have also been implicated in the pathogenesis of asthma. Patterns of cytokine production in the host may influence the pathogenesis of these diseases by regulating the activities of eosinophils and other components of the immune response. Mice which constitutively over-express IL-5 have profound and life-long eosinophilia in a restricted number of tissues. Although eosinophils from IL-5 transgenics are functionally competent for a number of parameters considered to be important in inflammation, untreated animals are overtly normal and free of disease. In addition, the responses of these animals when exposed to aeroallergens and helminths present a number of apparent paradoxes. Eosinophil accumulation in tissues adjacent to major airways is rapid and extensive in transgenics exposed to the aeroallergen, but even after treatment with antigen over many months these mice show no evidence of respiratory distress or pathology. Helminth-infected IL-5 transgenics and their non-transgenic littermates develop similar inflammatory responses at mucosal sites and are comparable for a number of T cell and antibody responses, but they differ considerably in their ability to clear some parasite species. The life-cycle of Nippostrongylus brasiliensis is significantly inhibited in IL-5 transgenics, but that of Toxocara canis is not. Our results also suggest that eosinophilia and/or over-expression of IL-5 may actually impair host resistance to Schistosoma mansoni and Trichinella spiralis. The pathogenesis of diseases in which eosinophils are involved may therefore be more complex than previously thought.

Central and Cortical Gray Mater Segmentation of Magnetic Resonance Images of the Fetal Brain

Motivation. The study of human brain development in itsearly stage is today possible thanks to in vivo fetalmagnetic resonance imaging (MRI) techniques. Aquantitative analysis of fetal cortical surfacerepresents a new approach which can be used as a markerof the cerebral maturation (as gyration) and also forstudying central nervous system pathologies [1]. However,this quantitative approach is a major challenge forseveral reasons. First, movement of the fetus inside theamniotic cavity requires very fast MRI sequences tominimize motion artifacts, resulting in a poor spatialresolution and/or lower SNR. Second, due to the ongoingmyelination and cortical maturation, the appearance ofthe developing brain differs very much from thehomogenous tissue types found in adults. Third, due tolow resolution, fetal MR images considerably suffer ofpartial volume (PV) effect, sometimes in large areas.Today extensive efforts are made to deal with thereconstruction of high resolution 3D fetal volumes[2,3,4] to cope with intra-volume motion and low SNR.However, few studies exist related to the automatedsegmentation of MR fetal imaging. [5] and [6] work on thesegmentation of specific areas of the fetal brain such asposterior fossa, brainstem or germinal matrix. Firstattempt for automated brain tissue segmentation has beenpresented in [7] and in our previous work [8]. Bothmethods apply the Expectation-Maximization Markov RandomField (EM-MRF) framework but contrary to [7] we do notneed from any anatomical atlas prior. Data set &Methods. Prenatal MR imaging was performed with a 1-Tsystem (GE Medical Systems, Milwaukee) using single shotfast spin echo (ssFSE) sequences (TR 7000 ms, TE 180 ms,FOV 40 x 40 cm, slice thickness 5.4mm, in plane spatialresolution 1.09mm). Each fetus has 6 axial volumes(around 15 slices per volume), each of them acquired inabout 1 min. Each volume is shifted by 1 mm with respectto the previous one. Gestational age (GA) ranges from 29to 32 weeks. Mother is under sedation. Each volume ismanually segmented to extract fetal brain fromsurrounding maternal tissues. Then, in-homogeneityintensity correction is performed using [9] and linearintensity normalization is performed to have intensityvalues that range from 0 to 255. Note that due tointra-tissue variability of developing brain someintensity variability still remains. For each fetus, ahigh spatial resolution image of isotropic voxel size of1.09 mm is created applying [2] and using B-splines forthe scattered data interpolation [10] (see Fig. 1). Then,basal ganglia (BS) segmentation is performed on thissuper reconstructed volume. Active contour framework witha Level Set (LS) implementation is used. Our LS follows aslightly different formulation from well-known Chan-Vese[11] formulation. In our case, the LS evolves forcing themean of the inside of the curve to be the mean intensityof basal ganglia. Moreover, we add local spatial priorthrough a probabilistic map created by fitting anellipsoid onto the basal ganglia region. Some userinteraction is needed to set the mean intensity of BG(green dots in Fig. 2) and the initial fitting points forthe probabilistic prior map (blue points in Fig. 2). Oncebasal ganglia are removed from the image, brain tissuesegmentation is performed as described in [8]. Results.The case study presented here has 29 weeks of GA. Thehigh resolution reconstructed volume is presented in Fig.1. The steps of BG segmentation are shown in Fig. 2.Overlap in comparison with manual segmentation isquantified by the Dice similarity index (DSI) equal to0.829 (values above 0.7 are considered a very goodagreement). Such BG segmentation has been applied on 3other subjects ranging for 29 to 32 GA and the DSI hasbeen of 0.856, 0.794 and 0.785. Our segmentation of theinner (red and blue contours) and outer cortical surface(green contour) is presented in Fig. 3. Finally, torefine the results we include our WM segmentation in theFreesurfer software [12] and some manual corrections toobtain Fig.4. Discussion. Precise cortical surfaceextraction of fetal brain is needed for quantitativestudies of early human brain development. Our workcombines the well known statistical classificationframework with the active contour segmentation forcentral gray mater extraction. A main advantage of thepresented procedure for fetal brain surface extraction isthat we do not include any spatial prior coming fromanatomical atlases. The results presented here arepreliminary but promising. Our efforts are now in testingsuch approach on a wider range of gestational ages thatwe will include in the final version of this work andstudying as well its generalization to different scannersand different type of MRI sequences. References. [1]Guibaud, Prenatal Diagnosis 29(4) (2009). [2] Rousseau,Acad. Rad. 13(9), 2006, [3] Jiang, IEEE TMI 2007. [4]Warfield IADB, MICCAI 2009. [5] Claude, IEEE Trans. Bio.Eng. 51(4) (2004). [6] Habas, MICCAI (Pt. 1) 2008. [7]Bertelsen, ISMRM 2009 [8] Bach Cuadra, IADB, MICCAI 2009.[9] Styner, IEEE TMI 19(39 (2000). [10] Lee, IEEE Trans.Visual. And Comp. Graph. 3(3), 1997, [11] Chan, IEEETrans. Img. Proc, 10(2), 2001 [12] Freesurfer,http://surfer.nmr.mgh.harvard.edu.

The effects of nutrition, puberty and dancing on bone density in adolescent ballet dancers.

Ballet dancers have on average a low bone mineral content (BMC), with elevated fracture-risk, low body mass index (BMI) for age (body mass index, kg/m2), low energy intake, and delayed puberty. This study aims at a better understanding of the interactions of these factors, especially with regard to nutrition. During a competition for pre-professional dancers we examined 127 female participants (60 Asians, 67 Caucasians). They averaged 16.7 years of age, started dancing at 5.8 years, and danced 22 hours/week. Assessments were made for BMI, BMC (DXA), and bone mineral apparent density (BMAD) at the lumbar spine and femoral neck, pubertal stage (Tanner score), and nutritional status (EAT-40 questionnaire and a qualitative three-day dietary record). BMI for age was found to be normal in only 42.5% of the dancers, while 15.7% had a more or less severe degree of thinness (12.6% Grade2 and 3.1% Grade 3 thinness). Menarche was late (13.9 years, range 11 to 16.8 years). Food intake, evaluated by number of consumed food portions, was below the recommendations for a normally active population in all food groups except animal proteins, where the intake was more than twice the recommended amount. In this population, with low BMI and intense exercise, BMC was low and associated with nutritional factors; dairy products had a positive and non-dairy proteins a negative influence. A positive correlation between BMAD and years since menarche confirmed the importance of exposure to estrogens and the negative impact of delayed puberty. Because of this and the probable negative influence of a high intake of non-dairy proteins, such as meat, fish, and eggs, and the positive association with a high dairy intake, ballet schools should promote balanced diets and normal weight and should recognize and help dancers avoid eating disorders and delayed puberty caused by extensive dancing and inadequate nutrition.

Double-oblique free-breathing high resolution three-dimensional coronary magnetic resonance angiography.

OBJECTIVES: The goal of the present study was to develop a strategy for three-dimensional (3D) volume acquisition along the major axes of the coronary arteries. BACKGROUND: For high-resolution 3D free-breathing coronary magnetic resonance angiography (MRA), coverage of the coronary artery tree may be limited due to excessive measurement times associated with large volume acquisitions. Planning the 3D volume along the major axis of the coronary vessels may help to overcome such limitations. METHODS: Fifteen healthy adult volunteers and seven patients with X-ray angiographically confirmed coronary artery disease underwent free-breathing navigator-gated and corrected 3D coronary MRA. For an accurate volume targeting of the high resolution scans, a three-point planscan software tool was applied. RESULTS: The average length of contiguously visualized left main and left anterior descending coronary artery was 81.8 +/- 13.9 mm in the healthy volunteers and 76.2 +/- 16.5 mm in the patients (p = NS). For the right coronary artery, a total length of 111.7 +/- 27.7 mm was found in the healthy volunteers and 79.3 +/- 4.6 mm in the patients (p = NS). Comparing coronary MRA and X-ray angiography, a good agreement of anatomy and pathology was found in the patients. CONCLUSIONS: Double-oblique submillimeter free-breathing coronary MRA allows depiction of extensive parts of the native coronary arteries. The results obtained in patients suggest that the method has the potential to be applied in broader prospective multicenter studies where coronary MRA is compared with X-ray angiography.

Genomics of "chlamydia"-related bacteria

AbstractThe Chlamydiales order is an important bacterial phylum that comprises some of the most successful human pathogens such as Chlamydia trachomatis, the leading infectious cause of blindness worldwide. Since some years, several new bacteria related to Chlamydia have been discovered in clinical or environmental samples and might represent emerging pathogens. The genome sequencing of classical Chlamydia has brought invaluable information on these obligate intracellular bacteria otherwise difficult to study due to the lack of tools to perform basic genetic manipulation. The recent emergence of high-throughput sequencing technologies yielding millions of reads in a short time lowered the costs of genome sequencing and thus represented a unique opportunity to study Chlamydia-re\ated bacteria. Based on the sequencing and the analysis of Chlamydiales genomes, this thesis provides significant insights into the genetic determinants of the intracellular lifestyle, the pathogenicity, the metabolism and the evolution of Chlamydia-related bacteria. A first approach showed the efficacy of rapid sequencing coupled to proteomics to identify immunogenic proteins. This method, particularly useful for an emerging pathogen such as Parachlamydia acanthamoebae, enabled us to discover good candidates for the development of diagnostic tools that would permit to evaluate at larger scale the role of this bacterium in disease. Second, the complete genome of Waddlia chondrophila, a potential agent of miscarriage, encodes numerous virulence factors to manipulate its host cell and resist to environmental stresses. The reconstruction of metabolic pathways showed that the bacterium possesses extensive capabilities compared to related organisms. However, it is still incapable of synthesizing some essential components and thus has to import them from its host. Third, the genome comparison of Protochlamydia naegleriophila to its closest known relative Protochlamydia amoebophila revealed a particular evolutionary dynamic with the occurrence of an unexpected genome rearrangement. Fourth, a phylogenetic analysis of P. acanthamoebae and Legionella drancourtii identified several genes probably exchanged by horizontal gene transfer with other intracellular bacteria that might occur within their amoebal host. These genes often encode mechanisms for resistance to metal or toxic compounds. As a whole, the analysis of the different genomes enabled us to highlight a large diversity in size, GC percentage, repeat content as well as plasmid organization. The abundant genomic data obtained during this thesis have a wide impact since they provide the necessary bases for detailed investigations on countless aspects of the biology and the evolution of Chlamydia-related bacteria, whether in wet lab or by bioinformatical analyses.RésuméL'ordre des Chlamydiales est un important phylum bactérien qui comprend de nombreuses espèces pathogènes pour l'homme et les animaux, dont Chlamydia trachomatis, responsable du trachome, la cause majeure de cécité d'origine infectieuse à travers le monde. Durant ces dernières décennies, de nombreuses bactéries apparentées aux Chlamydia ont été découvertes dans des échantillons environnementaux ou cliniques mais leur éventuel rôle pathogène dans le développement de maladies reste peu connu. Ces bactéries sont des intracellulaires obligatoires car elles ont besoin d'une cellule hôte pour se multiplier, ce qui rend leur étude particulièrement difficile. Le développement de nouvelles technologies permettant de séquencer le génome d'un organisme rapidement et à moindre coût ainsi que l'essor des méthodes d'analyse s'y rapportant représentent une opportunité exceptionnelle d'étudier ces organismes. Dans ce contexte, cette thèse démontre l'utilité de la génomique pour développer de nouveaux outils diagnostiques ainsi que pour étudier le métabolisme de ces bactéries, leurs facteurs de virulence et leur évolution.Ainsi, une première approche a illustré l'utilité d'un séquençage rapide pour obtenir les informations nécessaires à l'identification de protéines qui sont reconnues par des anticorps humains ou animaux. Cette méthode, particulièrement utile pour un pathogène émergent tel que Parachlamydia acanthamoebae, a permis de découvrir de bons candidats pour le développement d'un outil diagnostique qui permettrait d'évaluer à plus large échelle le rôle de cette bactérie notamment dans la pneumonie. L'analyse du contenu génique de Waddlia chondrophila, un autre germe qui pourrait être impliqué dans les avortements et tes fausses-couches, a en outre mis en évidence la présence de nombreux facteurs connus qui lui permettent de manipuler son hôte. Cette bactérie possède de plus grandes capacités métaboliques que les autres Chlamydia, mais elle est incapable de synthétiser certains composants et doit donc les importer de son hôte pour subvenir à ses besoins. La comparaison du génome de Protochlamydia naegleriophila à son plus proche parent, Protochlamydia amoebophila, a dévoilé une évolution dynamique particulière avec l'occurrence d'un réarrangement majeur inattendu après la séparation de ces deux espèces. En outre, ces études ont montré l'occurrence de plusieurs transferts de gène avec d'autres organismes plus éloignés, notamment d'autres intracellulaires d'amibes, souvent pour l'acquisition de mécanismes de résistances à des composés toxiques. Les données génomiques acquises durant ce travail posent les fondements nécessaires a de nombreuses analyses qui permettront progressivement de mieux comprendre de nombreux aspects de ces bactéries fascinantes.

Retrospective Study on Dengue in Fortaleza, State of Ceará, Brazil

A retrospective serologic study was carried out in Fortaleza, State of Ceará, Brazil, in order to detect the dengue virus activity before recognizing the epidemic of 1994. Mac-Elisa was performed by using a mixture of specific DEN-1 and DEN-2 antigens on serum samples from the Emilio Ribas Laboratory collection. Samples were obtained from 1,224 patients with exanthematic febrile disease and negative serological results for rubella. All specimens were taken during November 1993 to May 1994. The results confirmed dengue infections in Fortaleza by November 1993, approximately six months before the beginning of the epidemic, proving how misleading diagnosis of dengue infection are still troublesome, in spite of the strong dengue activity in Ceará. The authors stress the urgent necessity to implement the active surveillance system in order to prevent another extensive dengue fever epidemics in the state. Epidemiological background of the dengue activity in the State of Ceará is also described.

Cellular pathology of hilar neurons in Ammon's horn sclerosis.

In addition to functionally affected neuronal signaling pathways, altered axonal, dendritic, and synaptic morphology may contribute to hippocampal hyperexcitability in chronic mesial temporal lobe epilepsies (MTLE). The sclerotic hippocampus in Ammon's horn sclerosis (AHS)-associated MTLE, which shows segmental neuronal cell loss, axonal reorganization, and astrogliosis, would appear particularly susceptible to such changes. To characterize the cellular hippocampal pathology in MTLE, we have analyzed hilar neurons in surgical hippocampus specimens from patients with MTLE. Anatomically well-preserved hippocampal specimens from patients with AHS (n = 44) and from patients with focal temporal lesions (non-AHS; n = 20) were studied using confocal laser scanning microscopy (CFLSM) and electron microscopy (EM). Hippocampal samples from three tumor patients without chronic epilepsies and autopsy samples were used as controls. Using intracellular Lucifer Yellow injection and CFLSM, spiny pyramidal, multipolar, and mossy cells as well as non-spiny multipolar neurons have been identified as major hilar cell types in controls and lesion-associated MTLE specimens. In contrast, none of the hilar neurons from AHS specimens displayed a morphology reminiscent of mossy cells. In AHS, a major portion of the pyramidal and multipolar neurons showed extensive dendritic ramification and periodic nodular swellings of dendritic shafts. EM analysis confirmed the altered cellular morphology, with an accumulation of cytoskeletal filaments and increased numbers of mitochondria as the most prominent findings. To characterize cytoskeletal alterations in hilar neurons further, immunohistochemical reactions for neurofilament proteins (NFP), microtubule-associated proteins, and tau were performed. This analysis specifically identified large and atypical hilar neurons with an accumulation of low weight NFP. Our data demonstrate striking structural alterations in hilar neurons of patients with AHS compared with controls and non-sclerotic MTLE specimens. Such changes may develop during cellular reorganization in the epileptogenic hippocampus and are likely to contribute to the pathogenesis or maintenance of temporal lobe epilepsy.

Catabolic ornithine carbamoyltransferase of Pseudomonas aeruginosa. Importance of the N-terminal region for dodecameric structure and homotropic carbamoylphosphate cooperativity.

Pseudomonas aeruginosa has an anabolic (ArgF) and a catabolic (ArcB) ornithine carbamoyltransferase (OTCase). Despite extensive sequence similarities, these enzymes function unidirectionally in vivo. In the dodecameric catabolic OTCase, homotropic cooperativity for carbamoylphosphate strongly depresses the anabolic reaction; the residue Glu1O5 and the C-terminus are known to be essential for this cooperativity. When Glu1O5 and nine C-terminal amino acids of the catabolic OTCase were introduced, by in vitro genetic manipulation, into the closely related, trimeric, anabolic (ArgF) OTCase of Escherichia coli, the enzyme displayed Michaelis-Menten kinetics and no cooperativity was observed. This indicates that additional amino acid residues are required to produce homotropic cooperativity and a dodecameric assembly. To localize these residues, we constructed several hybrid enzymes by fusing, in vivo or in vitro, the E. coli argF gene to the P. aeruginosa arcB gene. A hybrid enzyme consisting of 101 N-terminal ArgF amino acids fused to 233 C-terminal ArcB residues and the reciprocal ArcB-ArgF hybrid were both trimers with little or no cooperativity. Replacing the seven N-terminal residues of the ArcB enzyme by the corresponding six residues of E. coli ArgF enzyme produced a dodecameric enzyme which showed a reduced affinity for carbamoylphosphate and an increase in homotropic cooperativity. Thus, the N-terminal amino acids of catabolic OTCase are important for interaction with carbamoylphosphate, but do not alone determine dodecameric assembly. Hybrid enzymes consisting of either 26 or 42 N-terminal ArgF amino acids and the corresponding C-terminal ArcB residues were both trimeric, yet they retained some homotropic cooperativity. Within the N-terminal ArcB region, a replacement of motif 28-33 by the corresponding ArgF segment destabilized the dodecameric structure and the enzyme existed in trimeric and dodecameric states, indicating that this region is important for dodecameric assembly. These findings were interpreted in the light of the three-dimensional structure of catabolic OTCase, which allows predictions about trimer-trimer interactions. Dodecameric assembly appears to require at least three regions: the N- and C-termini (which are close to each other in a monomer), residues 28-33 and residues 147-154. Dodecameric structure correlates with high carbamoylphosphate cooperativity and thermal stability, but some trimeric hybrid enzymes retain cooperativity, and the dodecameric Glu1O5-->Ala mutant gives hyperbolic carbamoylphosphate saturation, indicating that dodecameric structure is neither necessary nor sufficient to ensure cooperativity.

Characterization of the effects of IL-17F on CHO cell line generation

CHO is the most commonly used mammalian host for the generation of cell lines allowing for the production of high quality therapeutic proteins. The generation of such cell lines is a lengthy and resource-intensive process requiring extensive screening in order to isolate candidates with optimal characteristics, such as growth, stability and productivity. For this reason, the biotechnology industry invests much effort in attempts to optimize CHO expression systems in order to streamline and shorten the cell line selection process. Based on preliminary observations of a facilitated selection of CHO-GS cell lines expressing members of the IL-17 cytokine family, this study investigates the use of IL-17F as a novel enhancing factor for CHO cell line generation. Using two different CHO expression systems (exploiting GS and DHFR-based selection), we demonstrated that IL-17F expression caused a significant increase in the occurrence of colonies during the selection process. All colonies selected produced substantial amounts of IL-17F, suggesting that benefits were conferred, during selection, to those cells expressing the cytokine. Furthermore, transgene expression levels were significantly increased when the selection pressure was raised to a level that would not normally be permissive for colony selection (i.e. 100 |o.M MSX for the CHO-GS expression system or 1000 nM MTX for the CHO-DHFR system). Finally, IL-17F expression was also found to enhance the rate of appearance of clones during single cell subcloning in the absence of selection pressure. Overall, these benefits have the potential to allow a substantial reduction in the length of cell line generation while significantly increasing cell line productivity. Nevertheless, we found that the high IL-17F expression levels required to convey enhancing effects was a limitation when attempting to co-express IL-17F and a recombinant soluble protein of therapeutic interest from independent CMV promoters within the same expression vector. In order to understand and overcome this limitation, studies were designed to characterize the IL-17F enhancing effect at the molecular and cellular level. Regular supplementation of recombinant biologically-active IL-17F into the culture medium during cell line selection was not able to reproduce the enhancing effects of endogenous IL-17F expression. In addition, increased IL-17F expression correlated with increased CHO-GS selection transgene expression at the single cell level. This data suggested a possible effect of IL-17F on viral promoter activity or transgene mRNA stability. It also provided direct evidence that the cells expressing the highest amounts of IL-17F obtained the most benefit. Overall data obtained from these study implied that IL-17F may act through an intracellular mechanism, possibly exerted during secretion. We therefore initiated experiments designed to determine the specific compartment(s) within which IL-17F triggers its effect. This work has identified IL-17F as a potentially powerful tool to optimize the CHO cell line generation process. The characterization of this enhancing effect at the molecular level has given us several insights into overcoming the current limitations, thus paving the way for the development of a viable technology that can be exploited within the biotechnology industry. - La CHO est la cellule hôte de mammifere la plus couramment utilisée dans la création de lignée cellulaire produisant des protéines thérapeutiques de haute qualité. La génération de ces lignées cellulaires est un processus long et exigeant l'utilisation de techniques de sélection robustes afin d'isoler des candidats possédants les caractéristiques optimales de croissance, de productivité et de stabilité d'expression. Les industries biopharmaceutiques ont investi beaucoup d'efforts afin d'optimiser les systèmes d'expression CHO dans le but raccourcir la longueur du procédé de sélection de lignées cellulaires et aussi d'en augmenter l'efficacité. A partir d'observations préliminaires obtenues lors de la génération de lignées cellulaires CHO- GS exprimant une cytokine appartenant à la famille des IL-17, nous avons réalisé une étude portant sur l'utilisation de l'IL-17F humaine (IL-17F) comme nouveau facteur d'optimisation pour la génération de lignées cellulaires CHO. Nous avons démontré, en utilisant les deux systèmes de sélection et d'expression CHO couramment utilisés (le premier exploitant la GS et l'autre basée sur la DHFR), que l'expression de l'IL-17F permet une augmentation significative de la fréquence d'apparition de colonies durant le processus de sélection de lignées cellulaires. Les différentes colonies sélectionnées expriment des quantités substantielles d'IL-17F, suggérant un effet bénéfique lors de la sélection qui serait exclusivement conféré aux cellules exprimant la cytokine. En outre, le niveau d'expression du transgene se trouve significativement augmenté lorsque la pression de sélection est portée à un niveau habituellement trop élevé pour permettre la sélection de colonies (soit 100 |JM MSX pour le système d'expression CHO-GS ou 1000 nM MTX pour le système CHO- DHFR). Enfin, l'expression d'IL-17F permet également d'améliorer la vitesse d'apparition de clones pendant une étape de sous-clonage en l'absence de pression de sélection. L'ensemble de ces effets bénéfiques permettent une réduction substantielle de la durée de génération de lignées cellulaires tout en augmentant considérablement la productivité des lignées obtenues. Néanmoins, nous avons constaté que la nécessité d'exprimer des niveaux élevés d'IL-17F afin obtenir l'ensemble de ses effets bénéfiques devient une contrainte lors de l'utilisation d'un vecteur d'expression composé de deux promoteurs CMV indépendants pour la co-expression de la cytokine et d'une protéine soluble présentant un intérêt thérapeutique. Afin de mieux comprendre et de surmonter cette limitation, plusieurs études ont été effectuées dans le but de mieux caractériser l'effet de IL-17F au niveau subcellulaire. L'apport régulier en IL-17F recombinante et biologiquement active dans le milieu de culture lors de la sélection de lignées cellulaires ne permet pas de reproduire les effets bénéfiques observés par l'expression endogène d'IL-17F. En outre, nous avons constaté que, lors de l'utilisation du système CHO- GS, l'augmentation d'expression de 1TL-17F est corrélée à un accroissement de l'expression du marqueur de sélection au niveau cellulaire. Ces résultats suggèrent un possible effet d'IL- 17F sur l'activité des promoteurs viraux et ainsi fournissent une preuve directe que les cellules exprimant de haut niveau d'IL-17F sont celles qui en profitent le plus. L'ensemble de ces observations mettrait en avant que l'effet d'IL-17F se ferait selon un mécanisme intracellulaire. Nous avons donc étudié le(s) compartiment(s) spécifique(s) dans lequel IL-17F pourrait exercer son effet. Ce travail a permis de définir IL-17F comme un puissant outil pour l'optimisation des procédés de génération de lignées cellulaires CHO. La caractérisation de cette amélioration de l'effet au niveau moléculaire nous a donné plusieurs indications sur la manière de dépasser les limitations actuelles, ouvrant ainsi la voie au développement d'une technologie viable qui peut être exploitée pars l'industrie biotechnologique.