Optimization of a bioreactor type "soft-mixer" : for the growth of microalgae

Les toxines marines représentent une source très variée de molécules pour les nouvelles thérapies. Toutefois, elles sont actuellement très onéreuses car elles ne peuvent être produites en grande quantité. En effet, les micro-algues qui contiennent naturellement ces molécules, les dinoflagellés, ne peuvent pas être cultivées dans des photobioréacteurs (PBR) traditionnels car elles sont extrêmement sensibles au cisaillement. Dans cette thèse, nous expérimentons la culture de dinoflagellés dans un PBR innovant, le dinophyt, développé par l’IRPHE et la société Planktovie. Ce PBR, constitué d'un cylindre tournant autour de son axe, incliné par rapport à la verticale, permet d'apporter une grande quantité de CO2 aux microalgues avec des forces de cisaillement beaucoup plus faibles ( 100 mN.m-2). Dans une première partie, le flux de CO2 a été mesuré pour différentes vitesses de rotation. Le flux est inversement proportionnel à la racine carrée du nombre d'Ekman, ce qui s'explique par la présence de couches d'Ekman. Dans une deuxième partie, nous avons cultivé le dinoflagellé amphidinium carterae dans le dinophyt. En variant la vitesse de rotation, le renouvellement de milieu, le contrôle du pH et l’apport de CO2, nous avons montré que la culture d’A. carterae dans ce dinophyt est robuste par rapport à ces paramètres, ce qui rend son utilisation industrielle possible. Dans une troisième partie, des dinophyts de volumes industriels (> 100L) sont utilisés pour la culture d’A. carterae. Les rendements sont au moins 2 fois plus élevés que dans les PBR classiques, avec une production moyenne de 145 millions de cellules par litre par jourMarine toxins represent a very diverse source of molecules for new therapies.However, they are currently very expensive because they cannot be produced in largequantities. Indeed, the microalgae that naturally contain these molecules, the dinoflagellates, cannot be grown in traditional photobioreactors (PBRs) because they are extremely sensitive to shear. In this thesis, we experiment the culture of dinoflagellates in an innovative PBR, the dinophyt, developed by IRPHE and the company Planktovie. This PBR, consisting of acylinder rotating around its axis, inclined to the vertical, allows to bring a large amount of CO2 to the microalgae with much lower shear forces ( 100 mN.m-2). In a first part, the CO2 flux was measured for different rotation speeds. The flux is inversely proportional to the square root of the Ekman number, which is explained by the presence o fEkman layers. In a second part, we grew the dinoflagellate amphidinium carterae in dinophyt. By varying the rotation speed, medium renewal, pH control and CO2 supply, we showed that the culture of A. carterae in this Dinophyt is robust with respect to these parameters, which makes itsindustrial use possible.In a third part, dinophytes of industrial volumes (> 100L) are used for the culture of A.carterae. The yields are at least 2 times higher than in conventional PBRs, with an average production of 145 million cells per liter per da

Gas injection into a tilted rotating cylinder

The flux of CO 2 diffusing inside a new type of bioreactors has been measured experimentally. This geoinspired bioreactor consists in a partially filled cylinder rotating around its axis tilted with respect to the vertical, thus mimicking the precession motion of the Earth. The height of fluid is chosen equal to 2 radii in order for the first Kelvin mode to be resonant. The CO 2 diffuses through a membrane located at the bottom of the liquid. The partial pressure of CO 2 above the free surface is measured as a function of time. This temporal evolution is modeled by the presence of diffusive layers with no advection at the top and the bottom of the liquid. This basic model leads to an experimental value for the flux of CO 2 at the membrane, which is found to be proportional to the inverse square root of the Ekman number. This is in agreement with the presence of Ekman layers of thickness proportional to the square root of the Ekman number. At a given Ekman number the flux weakly depends on the tilt angle α with a scaling as α 1/4

Tilted rotating annular bioreactors

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Isolation of a Yersinia enterocolitica biotype 1B strain in France, and evaluation of its genetic relatedness to other European and North American biotype 1B strains.

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Towards a Common Mode Current Free Packaging Solution for High Voltage Series Connected SiC MOSFET Switches

International audienceTests are carried out on two different series-connected switches made of six SiC MOSFETs capable of blocking 10 kV and 20 kV respectively. High voltage capacitors are connected to the drain terminals of the MOSFETs to emulate the common mode currents' paths due to parasitic capacitances of the packaging. Due to high dV/dt, common mode currents become dangerous to the switch as they unsettle the voltage sharing during turn-off and unbalance switching losses. This could lead some of the MOSFETs to experience avalanche breakdown. A novel packaging approach that suppresses or balances the common mode currents is introduced

WWOX -related encephalopathies: delineation of the phenotypical spectrum and emerging genotype-phenotype correlation

International audienceBACKGROUND:Homozygous mutations in WWOX were reported in eight individuals of two families with autosomal recessive spinocerebellar ataxia type 12 and in two siblings with infantile epileptic encephalopathy (IEE), including one who deceased prior to DNA sampling.METHODS:By combining array comparative genomic hybridisation, targeted Sanger sequencing and next generation sequencing, we identified five further patients from four families with IEE due to biallelic alterations of WWOX.RESULTS:We identified eight deleterious WWOX alleles consisting in four deletions, a four base-pair frameshifting deletion, one missense and two nonsense mutations. Genotype-phenotype correlation emerges from the seven reported families. The phenotype in four patients carrying two predicted null alleles was characterised by (1) little if any psychomotor acquisitions, poor spontaneous motility and absent eye contact from birth, (2) pharmacoresistant epilepsy starting in the 1st weeks of life, (3) possible retinal degeneration, acquired microcephaly and premature death. This contrasted with the less severe autosomal recessive spinocerebellar ataxia type 12 phenotype due to hypomorphic alleles. In line with this correlation, the phenotype in two siblings carrying a null allele and a missense mutation was intermediate.CONCLUSIONS:Our results obtained by a combination of different molecular techniques undoubtedly incriminate WWOX as a gene for recessive IEE and illustrate the usefulness of high throughput data mining for the identification of genes for rare autosomal recessive disorders. The structure of the WWOX locus encompassing the FRA16D fragile site might explain why constitutive deletions are recurrently reported in genetic databases, suggesting that WWOX-related encephalopathies, although likely rare, may not be exceptional

Clinical study of 19 patients with SCN 8A ‐related epilepsy: Two modes of onset regarding EEG and seizures

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Molecular and clinical description of patients with GABA A receptor gene variants ( GABRA1 , GABRB2 , GABRB3 , GABRG2 ): a cohort study, review of literature, and genotype‐phenotype correlations

International audienceObjective: GABAA receptor subunit variants have recently been associated with neurodevelopmental disorders and/or epilepsy. The phenotype linked with each gene is becoming better known. Because of their common molecular structure and physiological role, it seemed interesting to describe a putative phenotype associated with GABAA -receptor-related disorders as a whole and seek possible genotype/phenotype correlations.Methods: We collected clinical, electrophysiological, therapeutic, and molecular data from patients affected with GABAA receptor subunit variants (GABRA1, GABRB2, GABRB3, GABRG2) through a national French collaboration using the EPIGENE network and compared them to the one already described in the literature.Results: We gathered the reported patients in 3 epileptic phenotypes: 15 patients with fever-related epilepsy (40%), 11 with early developmental epileptic encephalopathy (30%), 10 with generalized epilepsy spectrum (27%), and 1 patient without seizures (3%). We did not find a specific phenotype for any gene, but we showed that the location of variants on the transmembrane (TM) segment was associated with a more severe phenotype, irrespective of the GABAA receptor subunit gene, whereas N-terminal variants seemed to be related to milder phenotypes.Significance: GABAA receptor subunit variants are associated with highly variable phenotypes despite their molecular and physiological proximity. None of the genes described here was associated with a specific phenotype. On the other hand, it appears that the location of the variant on the protein may be a marker of severity. Variant location may have important weight in the development of targeted therapeutics

Gain-of-function and loss-of-function variants in GRIA3 lead to distinct neurodevelopmental phenotypes

International audienceAbstract AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors (AMPARs) mediate fast excitatory neurotransmission in the brain. AMPARs form by homo- or heteromeric assembly of subunits encoded by the GRIA1-GRIA4 genes, of which only GRIA3 is X-chromosomal. Increasing numbers of GRIA3 missense variants are reported in patients with neurodevelopmental disorders (NDD), but only a few have been examined functionally. Here, we evaluated the impact on AMPAR function of one frameshift and 43 rare missense GRIA3 variants identified in patients with NDD by electrophysiological assays. Thirty-one variants alter receptor function and show loss-of-function (LoF) or gain-of-function (GoF) properties, whereas 13 appeared neutral. We collected detailed clinical data from 25 patients (from 23 families) harbouring 17 of these variants. All patients had global developmental impairment, mostly moderate (9/25) or severe (12/25). Twelve patients had seizures, including focal motor (6/12), unknown onset motor (4/12), focal impaired awareness (1/12), (atypical) absence (2/12), myoclonic (5/12), and generalized tonic-clonic (1/12) or atonic (1/12) seizures. The epilepsy syndrome was classified as developmental and epileptic encephalopathy in eight patients, developmental encephalopathy without seizures in 13 patients, and intellectual disability with epilepsy in four patients. Limb muscular hypotonia was reported in 13/25, and hypertonia in 10/25. Movement disorders were reported in 14/25, with hyperekplexia or non-epileptic erratic myoclonus being the most prevalent feature (8/25). Correlating receptor functional phenotype with clinical features revealed clinical features for GRIA3-associated NDDs and distinct NDD phenotypes for LoF and GoF variants. GoF variants were associated with more severe outcomes: patients were younger at the time of seizure onset (median age one month), hypertonic, and more often had movement disorders, including hyperekplexia. Patients with LoF variants were older at the time of seizure onset (median age 16 months), hypotonic, and had sleeping disturbances. LoF and GoF variants were disease-causing in both sexes but affected males often carried de novo or hemizygous LoF variants inherited from healthy mothers, whereas all but one affected females had de novo heterozygous GoF variants