Foi realizado o pirossequenciamento de duas bibliotecas de cDNA do intestino médio de Tenebrio molitor e as sequências foram submetidos à montagem através do programa Newbler. Visando sanar alguns questionamentos a respeito de muitos tipos de transportadores que pudessem estar envolvidos com funções presumíveis em tamponamento luminal, absorção de nutrientes, envolvimento em mecanismos de secreção de enzimas como a α-manosidases e secreção e absorção de água, foram analisadas sequências de interesse que pudessem esclarecer os fenômenos fisiológicos em questão. O pirossequenciamento revelou 19 sequencias de α-manosidases. Após alinhamentos múltiplos, desconfiou-se que o contig 12 era a continuação da sequência original da α-manosidase. Utilizando-se de iniciadores apropriados, a suspeita foi confirmada e uma sequência completa foi obtida e denominada de TmMan1. Através de cladogramas gerados com as sequências de todos os contigs obtidos, assim como de sequências representativas das famílias 38 e 47 das glicosídeos hidrolases, mostrou que todas a nossas sequências, exceto o contig 6 e 7, pertencem à família 38. Todas as sequências com mais de 100 reads (exceto o contig 9) tiveram a sua expressão tecidual avaliada por RT-PCR. Os resultados mostraram que só são expressos no intestino ou intestino e túbulo de Malpighi, implicando na possibilidade de serem digestivas. Dessas sequências, as únicas com peptídeo sinal são a TmMan1 (contig 12) e o contig 14 e, portanto, devem corresponder às atividades Man1 e Man2. Levando em conta o número de reads, TmMan1 deveria corresponder a Man2 e o contig 14 à Man1. É possível, embora necessite de confirmação, que os contigs 8 e 15 sejam de expressão lisossômica. Um peptídeo sintetizado que correspondia a sequencia única da TmMan1 foi usado para gerar anticorpos, que reconheceram a Man2, mas não a Man1, confirmando a identificação de TmMan1 com a Man2. Esse anticorpo foi também utilizado para imunolocalizar a TmMan1 nas células intestinais de T. molitor. Os resultados mostraram que a TmMan1 é secretada de forma apócrina pela região anterior de intestino de T. molitor. Esse trabalho é o primeiro que mostra a ocorrência de α-manosidases com especificidade similar àquelas lissossômicas, mas que são secretadas apócrinamente para fora da célula, devendo agir no lúmen intestinal, removendo resíduos de manoses de oligossacarídeos manosilados. Foram identificados 10 tipos diferentes de transportadores e na elaboração dos modelos fisiológicos só foram levados em conta aqueles expressos exclusivamente no intestino médio ou no intestino médio e túbulos de Malpighi. A V-ATPase em T.molitor parece ser uma bomba usada para energizar muitos dos transportes ao longo do intestino médio como, por exemplo, o de oligopeptídeos. Já as bombas de Na+ e K+ são responsáveis pelo equilíbrio de cargas e, portanto estão presentes na maioria dos tipos celulares. Duas sequências de cotransportadores de oligopeptídeos/H+ foram encontradas no pirossequenciamento e sua expressão é maior na região posterior, uma vez que ali é a última possibilidade de absorção dos oligopeptídeos que ainda estiverem no lúmen, remanescentes da digestão final de proteínas. Foi demonstrado que T.Molitor absorve aminoácidos e açúcares ao longo de todo o intestino médio, pois estes tipos de transportadores possuem uma expressão uniforme ao longo do intestino médio. Já a expressão dos transportadores de NH3/NH4+ em T.molitor, encontra-se confinada ás regiões onde o pH do intestino médio do inseto é mais ácida. Também há uma expressão de transportadores de cloreto que se manifesta mais intensamente na região anterior. Podemos visualizar que a distribuição dos contigs dos transportadores de bicarbonato encontra-se mais expressiva na região posterior do intestino médio. Os resultados sugerem que a acidificação na região anterior do intestino de T.molitor pode resultar da secreção de NH4+ acompanhado do íon cloreto e a alcalinização na região posterior do lançamento no lúmen de bicarbonato. A importância dos canais de cloreto é que o mesmo balanceia as cargas e desta forma pode ser útil juntamente com o transporte de NH4+, que gera uma carga no lado onde é transportado. Há absorvição de água (junto com glicose) ao longo de todo o intestino médio, enquanto que a secreção de água ocorreria apenas nos dois terços finais do intestino médio com o auxílio de aquaporinas complementado por transportadores de íons, teria como consequência a abosorção líquida de água na região anterior e uma secreção líquida no final do intestino médio. Isso esclarece qual a base molecular para a ocorrência do contrafluxo intestinal evidenciado por experimentos fisiológicos. / Pyrosequencing was performed with two cDNA libraries in the midgut of Tenebrio molitor and the sequences were subjected to assembly with the Newbler program. In order to tackle questions concerning proteins which may be involved in midgut buffering, nutrient absorption, in the secretion of enzymes such as α-mannosidases , and water absorption and secretion, sequences of interest were analyzed in order to clarify those physiological phenomena. The pyrosequencing revealed 19 sequences of α- mannosidases . After multiple alignments, it was suspected that the contig 12 was the continuation of the original sequence of the α-mannosidase. Using appropriate primers, the hypothesis was confirmed and a complete sequence was obtained and named TmMan . Through cladograms generated from the sequences of the contigs obtained, as well as of sequences representing families 38 and 47 of glycoside hydrolases, it was showed that all sequences except the contig 6 and 7 belong to family 38. All sequences with over 100 reads (except contig 9) had their tissue expression assessed by RT-PCR. The results showed that they are expressed only in the midgut or midgut and Malpighian tubules, implying the possibility of having a digestive function. Among these sequences, the only ones with a signal peptide are TmMan1 (contig 12) and contig 14 and therefore they should correspond to the activities Man1 and Man2. Taking into account the number of reads, TmMan1 should correspond to Man2 and contig 14 to Man1. It is possible, though it requires confirmation, that the contigs 8 and 15 are lysosomal. A peptide corresponding to the unique sequence TmMan1 was synthesized and used to generate antibodies that recognized Man2 , but not Man1, confirming the identification of TmMan1 with Man2. This antibody was also used to immunolocalyze TmMan1 in the midgut cells of T. molitor. The results showed that TmMan1 is secreted in an apocrine way by the anterior region of T. molitor midgut. This is the first study that shows the occurrence of α-mannosidases with similar specificity to those lysosomal, but that are secreted in an apocrine way, acting in the midgut lumen, removing mannoses from mannosylated oligosaccharides. We identified 10 different types of carriers and in the development of physiological models it was only taken into account those expressed exclusively in the midgut or midgut and Malpighian tubules . The V- ATPase in T.molitor appears to be a pump used for powering many transports along the midgut, as of oligopeptides. The Na+ and K+ pumps are responsible for charge load balancing and therefore are present in most cell types. Two sequences of oligopeptide / H+ cotransporters were found in the transcriptome and their expression is higher in the posterior region. This agrees with the fact that there is the last possibility of oligopeptides remaining in the lumen to be absorbed. It is highly probable that T.molitor absorbs amino acids and sugars throughout the midgut, once these types of carriers have a uniform expression throughout the midgut . The expression of NH3/NH4+ transporters in T.molitor is confined to the regions where the pH of the insect midgut is more acidic. There is also chloride transporter expression there. The expression of bicarbonate transporters is more significant in the posterior midgut. The results suggest that acidification in the anterior T.molitor midgut may result from the secretion of NH4+ and chloride ions together, whereas the alkalization in the posterior midgut results from bicarbonate release. There is water absortion (along with glucose) throughout the midgut , while the water secretion occurs only in the final two-thirds of the midgut with the aid of aquaporins, complemented by ion transporters. It would result in the net absorption and net secretion of water in the anterior and postertior midgut, respectively. This clarifies the molecular basis of the midgut countercurrent fluxes evidenced by physiological experiments.

Orientador: Andréa Balan Fernandes / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-17T16:02:06Z (GMT). No. of bitstreams: 1 Pegos_VanessaRodrigues_M.pdf: 19133775 bytes, checksum: abcda42c9097c7e59ea61175934c2304 (MD5) Previous issue date: 2011 / Resumo: A captação de sulfato em Escherichia coli é dependente do transportador do tipo ABC (do inglês, ATP Binding Cassete), SbpCysAWD, pertencente a um regulon que envolve 26 genes. Na ausência de sulfato, a bactéria induz a expressão de dois outros transportadores ABC, o de proteínas do sistema de transporte de alcanosulfonatos (SsuABCDE) e de sulfonatos alifáticos (TauABCDE), que são responsáveis pela captação, incorporação e oxidação destes compostos a sulfito e aldeído. Embora não comprovado funcionalmente, Xanthomonas axonopodis pv. citri apresenta todos os genes envolvidos neste regulon e, neste trabalho, dando continuidade à caracterização do transportador SsuABCDE, foi realizada pela primeira vez, a caracterização estrutural e funcional das enzimas SsuD e SsuE. Análises bioquímicas associadas às análises de bioinformática e modelagem molecular, revelaram que as enzimas SsuD e SsuE constituem um sistema de dois componentes, no qual a SsuD seria a óxido-redutase responsável pela oxidação do NAD(P)H, seguida da redução da FMN. A proteína foi expressa e purificada a partir de células de E. coli com massa molecular de 39 kDa, e se organiza na forma de um octâmero, conforme demonstrado por experimentos de espalhamento de raios X a baixo ângulo e ultra-centrifugação. O modelo da estrutura tridimensional da SsuD revela uma proteína com enovelamento barril-TIM, com conservação de resíduos que permitem a oligomerização e a interação com NAD(P)H, mas não com flavina. Ensaios enzimáticos mostraram que a SsuD liga NADP(Pbp) com alta afinidade (0,21 µM) e é capaz de oxidá-lo conforme os parâmetros cinéticos de Km: 0.1877 µM -1; Kcat: 7,192 s-1; Vmáx: 0.7911 µM/min; Kcat/Km: 3,8 x 107 m-1S-1. Ainda, foram obtidos cristais da SsuD em diferentes condições as quais estão em fase de refinamento. As análises de bioinformática da SsuE sugerem que ela seja a óxido-redutase do sistema. O trabalho ainda mostra a caracterização da proteína Pbp de X. axonopodis, do suposto sistema de transporte de fosfonatos. A Pbp foi expressa com massa molecular de 33 kDa, e as análises espectroscópicas revelaram alterações conformacionais na estrutura secundária na presença de fosfonatos e fosfato, bem como aumentada estabilidade térmica na presença de espermidina, usada nos ensaios de cristalização. Cristais foram obtidos em diferentes condições e devem ser usados para os testes de difração. Os resultados apresentados neste trabalho revelam dados de proteínas e sistemas de X. axonopodis ainda não estudados e serão importantes para direcionar futuros estudos sobre a função destas na bactéria, tanto em condições laboratoriais, como em testes in vivo, durante infecção na planta / Abstract: Sulfur uptake in Escherichia coli is dependent of the ABC transporter SbpCysAWD (ATP Binding Cassete), which belongs to a regulon with 26 genes. In the sulfate absence, the bacteria induces the expression of two other ABC transporters, for alkanesulfonates (SsuABCDE) and aliphatic sulfonates (TauABCDE), which are responsible for the uptake and oxidation of these compounds to sulfite and aldehyde. Although its functionality has been not showed, Xanthomonas axonopodis pv. citri has all the genes involved in this regulon, including the alkanesulfonate transporter and enzymes. In order to continue the characterization of this transport, this work shows for the first time, the structural and functional characterization of the proteins SsuD and SsuE. Biochemical analyses associated to the bioinformatics and molecular modeling tools revealed that the SsuD and SsuE form a two-components system, where SsuD is the oxidoreductase responsible for the NAD(P)H oxidation followed by the FMN reduction. The protein was expressed and purified from E. coli cells with a molecular mass of the 39 kDa and it is organized in a octamer, such was demonstrated by the small angle scattering X-ray and ultracentrifugation assays. The tri-dimensional model of SsuD reveals a TIM barrel folding and conservation of residues that allow the oligomerization and NADP interaction. Enzymatic assays showed a high affinity binding of SsuD and NADP (0,21 µM) and that the protein was able to obtain the cinetic parameters, such as Km: 0.1877µM -1; Kcat: 7,192 s-1; Vmáx: 0.7911 µM/min; Kcat/Km: 3,8x 10-7 m 1S-1. Indeed, SsuD crystals were obtained in different conditions. The work still shows the charactrization of the Pbp protein, from X. axonopodis, believed to be the fosfonate/fosfate binding protein. Pbp was expressed with a molecular mass of 33 kDa, and spectroscopic analyses revealed conformational changes at the secondary structure content in presence of fosfonates, as well as an increased thermal stability in presence of spermidine, which was used for the crystallization trials. Crystals were obtained but still not tested. All results presented in this work can bring some light to the strategies that X. axonopodis uses for growth and infection and they will direct our studies for laboratorial and in vivo analyses / Mestrado / Genetica de Microorganismos / Mestre em Genética e Biologia Molecular

Mytilus edulis est un mollusque bivalve de grand intérêt économique et écotoxicologique. Cette espèce sentinelle est connue pour sa résistance aux contaminants chimiques et biologiques. Néanmoins, depuis quelques années la moule bleue est touchée par des mortalités dans les élevages des Pertuis Charentais ayant pour dénominateur commun la présence de bactéries virulentes de type Vibrio. Le premier axe de cette thèse décrit les interactions des isolats de V. Splendidus avec la moule bleue au niveau cellulaire et physiologique. Les infections expérimentales ont permis la sélection de deux isolats bactériens affiliés à V. splendidus/V. hemicentroti : une souche virulente codée 10/068 1T1 et une souche inoffensive codée 12/056 M24T1. Ces deux bactéries ont été marquées à la GFP et validées en tant que modèles authentiques d’exposition à travers leurs caractéristiques de croissance et de virulence. Par ailleurs, V. hemicentroti 10/068 1T1 est capable d’altérer différentes fonctions hémocytaires incluant la motilité, l’adhésion, l’internalisation, la production de ROS, la maturation du phagosome et la viabilité contrairement à la bactérie non virulente. Les produits extracellulaires bactériens semblent être toxiques et inhibent certaines réponses cellulaires (internalisation et production de ROS). Enfin, nous avons reproduis avec succès l’infection des animaux par le pathogène via un modèle expérimental de cohabitation. Le suivi de l’infection montre que V. hemicentroti 10/068 1T1 a pour cible principale les branchies. Le deuxième axe explore le fonctionnement du système MXR (MultiXenobiotic Resistance) chez la moule bleue. La séquence codante complète d’un nouveau transporteur ABCG2 a été établie et la protéine résultante a été identifiée. La caractérisation moléculaire montre la présence du transcrit dans les hémocytes ainsi que dans les branchies et son homologie avec les autres protéines appartenant à diverses espèces. L’utilisation des sondes fluorescentes bodipy prazosin et pheophorbide A, combinées avec des bloqueurs spécifiques, démontre l’activité d’efflux de ce transporteur et son hétérogénéité dans les tissus et cellules. Par ailleurs, il est également démontré que l’expression des trois transporteurs ABC (abcb, abcc, abcg2) identifiés chez la moule bleue est modulée par les contaminants chimiques. Les animaux exposés au BaP au laboratoire ou prélevés sur un terrain contaminé montrent une surexpression des transcrits abc dans les branchies et une sous expression dans les hémocytes. La saisonnalité, sur le terrain, a également un effet sur les niveaux des transcrits et interfère avec les réponses liées aux contaminants. Seul le transporteur abcb exprimé dans les branchies n’est pas affecté par des variations saisonnières et montre une surexpression dans le site contaminé tout au long de l’année. En conclusion, nos résultats démontrent la vulnérabilité de la moule bleue face un pathogène. L'impact immunotoxique des xénobiotiques et le rôle que peuvent jouer les transporteurs ABC dans le fonctionnement du système immunitaire des moules reste à explorer. / Mytilus edulis is a bivalve mollusc representing an economic and ecotoxicological interest. This sentinel species is known for its resistance to chemical and biological contaminants. However, for few years, abnormal mortality events have been reported for farmed blue mussels in France where different Vibrio strains were isolated. The first section of this thesis describes cellular and physiological interactions of V. Splendidus isolates with the blue mussel. Experimental infections allowed the selection of two isolates affiliated to V. splendidus/V. hemicentroti type strains: a virulent 10/068 1T1 and an innocuous 12/056 M24T1. These two strains were GFP-tagged and validated for their growth characteristics and virulence as genuine models for exposure. V. splendidus 10/068 1T1 is capable to alter different functions of hemocytes including motility, adhesion, internalization, ROS production, phagosome maturation and viability, unlike the avirulent strain. Furthermore, bacterial extracellular products appeared toxic and inhibit cellular responses (internalization and ROS production). Finally, we successfully reproduced experimental infection by water tank cohabitation assays with septic animals. Infection monitoring shows the targeting of gills by bacteria. The second section explores the MXR (MultiXenobiotic Resistance) system functioning in the blue mussel. For the first time, a complete ABCG2 amino acid sequence was established. Molecular characterization shows the presence of the abcg2 transcript in hemocytes and gills and its homology with other proteins from various species. The combination of the fluorescent probes bodipy prazosin and pheophorbide A with specific blockers demonstrate the transporter efflux activity and its heterogeneity in tissues and cells. Moreover, the expression of three ABC transporters (abcb, abcc, abcg2) identified in the blue mussel has been shown to be modulated by chemical contaminants. Mussels exposed to BaP in the laboratory or collected from contaminated mussel beds in the field show upregulated abc transcripts in gills whereas these mRNA undergone a downregulation in hemocytes. Season had also an effect on mRNA levels and interacted with site effects. Only the abcb gene displayed a more abundant mRNA level in gills dissected from animals collected in the more polluted area all over the diachronic study. In conclusion, our results demonstrate the vulnerability of the blue mussel towards a pathogen. The immunotoxic impact of environmental xenobiotics and the precise role of ABC efflux pumps in the immune system of the mussel has yet to be explored.

L'effet stimulateur de la croissance et de la nutrition des plantes exercés par les PGPR (Plant Growth-Promoting Rhizobacteria) a longtemps été étudié en s'intéressant à la bactérie. Cependant, les voies de signalisations impliquées dans la réponse de la plante à l'inoculation restent mal étudiées. A cet effet, notre étude entre dans le cadre des recherches visant les réponses physiologiques et moléculaires de la plante induites par une PGPR. Dans notre équipe de recherche, nous avons choisi la PGPR Phyllobacterium brassicacearum STM196 isolée de la rhizosphère de Colza et nous l'avons inoculée à la plante modèle Arabidopsis thaliana. Cette PGPR a montré sa capacité à stimuler l'allongement des racines latérales et des poils racinaires ainsi que d'augmenter la production de biomasse par la plante. Une forte surexpression de deux gènes de la famille de transporteurs de nitrate NRT2, NRT2.5 et NRT2.6, a été observée chez les plantes inoculées avec STM196. La fonction des produits de ces deux gènes n'est pas connue. Cependant, les données de transcriptomiques accumulées dans l'équipe font ressortir ces deux gènes comme des candidats intéressants dans les réponses moléculaires à l'interaction avec STM196. D'autre part, des études précédentes dans l'équipe ayant montré des effets antagonistes de la bactérie et du nitrate sur le développement racinaire, il est important de considérer la relation entre les effets de la nutrition nitrique et de la bactérie. Le principal transporteur responsable de l'absorption de NO3- étant NRT2.1, nous nous sommes intéressés à son rôle dans les réponses de la plante à la bactérie et à sa relation éventuelle avec NRT2.5 et NRT2.6. Nous avons réalisé une approche de génomique inverse avec les trois simples mutants ko nrt2.1, ko nrt2.5 et ko nrt2.6 dont nous disposions au départ, et avec les trois doubles mutants nrt2.5xnrt2.6, nrt2.1xnrt2.6 et nrt2.1xnrt2.5 que nous avons généré. Nous avons démontré que les gènes NRT2.5 et NRT2.6 sont impliqués dans les réponses de stimulation de croissance de la plante et de modification d'architecture racinaire à la PGPR STM196. Cette voie de régulation est indépendante des contrôles exercés par le statut azoté de la plante.Mots clés: Interaction plante-microorganisme, Phyllobacterium brassicacearum STM196, Arabidopsis thaliana, transporteurs de nitrate, NRT2.1, NRT2.5, NRT2.6, Activité nitrate réductase, NR1, expression des gènes. / AbstractThe promotion of plant growth and nutrition by some rhizospheric bacteria (Plant Growth Promoting Rhizobacteria, PGPR) is well known for a long time. However, the signaling pathways involved in the plant responses to these bacteria still remain essentially obscure. Our study aims at identifying molecular factors of plant physiological and developmental responses induced by PGPR. For this goal, we used the PGPR strain Phyllobacterium brassicacearum STM196, which has been isolated from rape rhizosphere, and the plant model Arabidopsis thaliana. This PGPR stimulates lateral root and root hair elongation and induce an increase of plant biomass production. Two genes of the NRT2 family of nitrate transporters, namely NRT2.5 and NRT2.6, are strongly overexpressed upon inoculation of Arabidopsis with STM196. The function of NRT2.5 and NRT2.6 is not known. However, transcriptomic data obtained in our team show that these two genes are promising candidates of the molecular responses to STM196. In addition, previous work in our team showed antagonistic effects of STM196 and exogenous nitrate on root development, showing that the effects of the bacteria must be considered together with those of nitrate nutrition. Since NRT2.1 is the major transporter for NO3- uptake, we looked at its role in the plant response to STM196 and its possible relationship with NRT2.5 and NRT2.6. We carried out a reverse genetic approach using the single mutants ko nrt2.1, ko nrt2.6 and ko nrt2.5 available at the moment this thesis work began and the double mutants nrt2.5xnrt2.6, nrt2.1xnrt2.6 and nrt2.1xnrt2.5 we generated. We demonstrated that NRT2.5 and NRT2.6 are involved in plant growth stimulation by STM196 and the root architecture changes elicited by this bacterium. This NRT2.5/NRT2.6-dependent pathway is independent from the regulations exerted by N nutritional status. Key words: Plant-microorganism interaction, Phyllobacterium brassicacearum STM196, Arabidopsis thaliana, nitrate transporter, NRT2.1, NRT2.5, NRT2.6, nitrate reductase activity, NR1, genes expression.

La survenue de lésions hépatiques représente une cause majeure de retrait des médicaments au cours de leur développement et après leur mise sur le marché. La manifestation la plus fréquente des effets secondaires liés aux médicaments est lacholestase qui résulte d’un blocage de la sécrétion biliaire. Ils peuvent être prévisibles, généralement dépendants de la dose, ou dans certains cas n’être observés que chez un nombre restreint de patients traités, par exemple avec la chlorpromazine (CPZ), un neuroleptique. Il s’agit alors d’une hépatotoxicité idiosyncratique. Notre travail a eu pour but d’induire une cholestase avec ce médicament et d’étudier les mécanismes impliqués, en présence ou non d’un stress inflammatoire en utilisant comme modèle expérimental les cellules hépatiques différenciées HepaRG dérivées d’un cholangio-hépatocarcinome humain. Nous avons tout d’abord validé ce modèle en montrant que les principaux transporteurs d’influx et d’efflux canaliculaires et basolatéraux sont bien localizes dans les domaines membranaires appropriés, et que les canalicules biliaires sont fonctionnels et fermés comme dans les hépatocytes humains cultivés en sandwich, le modèle de référence. Le traitement par CPZ à une concentration élevée (50μM) entraine après 15min la génération d’un stress oxydant associé à une altération du potentiel membranaire mitochondrial et de la distribution péricanaliculaire des microfilaments de F-actine et à une inhibition de l’efflux canaliculaire de l’acide taurocholique. Après 24h, on observe notamment une inhibition de l’expression des deux principaux transporteurs canaliculaires, BSEP et MDR3, du transporter d’influx NTCP et une surexpression du transporteur basolatéral MRP4. Ces effets suggèrent une réponse compensatrice des cellules face à l’accumulation intracellulaire des acides biliaires. L’inflammation est considérée comme un facteur de susceptibilité dans l’hépatotoxicité idiosyncratique. Nous avons recherché si dans un context inflammatoire induit par l’IL-6 et l’IL-1β, les effets cytotoxiques et cholestatiques de CPZ sont aggravés. Après un prétraitement de 24h par les deux cytokines proinflammatoires, les cellules HepaRG, ont été co-exposées à 20μM CPZ pendant 1 à 5 jours. Bien que les cytokines aient induit un stress inflammatoire et inhibé le métabolisme de la CPZ et les transcrits de CYP3A4 et CYP1A2, deux principaux CYPs impliqués dans le métabolisme de ce médicament, la modulation des effets cytotoxiques et cholestatiques de la CPZ observés est restée limitée, y compris après 5 jours. Une cytotoxicité accrue de 20% et une amplification de l’inhibition des transcrits et de l’activité de NTCP ainsi que la dérégulation de l’expression d’autres gènes liés à la cholestase, ont été constatées suite au co-traitement à CPZ et aux cytokines. Au total, nos résultats montrent qu’il est possible d’induire une cholestase in vitro à partir des cellules HepaRG et que la cholestase induite par CPZ a pour origine l’induction d’un stress oxydant. Ils montrent en outre que l’étude de certains facteurs de susceptibilité peut être envisagée. / Drug-induced liver injury is the major cause of drug withdrawal during development and marketing process. The most common manifestation of adverse drug reactions is cholestasis, which results from alteration of bile flow. Adverse drug reactions are usually classified either as dose-dependent and reproducible (intrinsic) or unpredictable (idiosyncratic) occurring only in certain susceptible patients as observed with chlorpromazine (CPZ), a neuroleptic drug. Our work aimed to induce cholestasis with this drug and to study the mechanisms involved in the presence or absence of an inflammatory stress using differentiated HepaRG liver cells derived from a human cholangio-hepatocarcinoma as an experimental model. We firstly validated this cell model by demonstrating that the major canalicular and basolateral influx and efflux transporters are localized to the appropriate membrane domains, and that the bile canaliculi are functional and closed as in sandwichcultured human hepatocytes, the reference model. Treatment with CPZ at a high concentration (50μM) induces, as early as 15min, generation of oxidative stress which is associated with altered mitochondrial membrane potential, disruption of the pericanalicular F-actin cytoskeleton distribution and inhibition of canalicular efflux of taurocholic acid. After 24-hour treatment with CPZ, mRNA expression of the two main canalicular bile transporters, BSEP and MDR3, and of the main influx transporter, NTCP, was decreased. By contrast, expression of MRP4 mRNA, a basolateral transporter, was increased. These latter events likely represent hepatoprotective responses which aim to reduce intrahepatic accumulation of toxic BA. Inflammation is considered as a factor of susceptibility to idiosyncratic hepatotoxicity. We investigated whether in an inflammatory stress induced by IL-6 and IL-1β, cytotoxic and cholestatic effects of CPZ are exacerbated. After a 24 hour pre-treatment by either pro-inflammatory cytokines, HepaRG cells were co-exposed to 20μM CPZ for 1 to 5 days. Although cytokines have induced inflammatory stress and inhibited the metabolism of CPZ and transcripts of CYP3A4 and CYP1A2, two main CYPs involved in the metabolism of this drug, the modulation of cytotoxic and cholestatic effects of CPZ was limited, even after 5 daily treatments. Increased cytotoxicity by 20 %, amplification of NTCP mRNA and activity inhibition and deregulation of the expression of other genes associated with cholestasis, were observed in CPZ- and cytokine-co-treated cells. Altogether, our results show that it is possible to induce in vitro cholestasis using HepaRG cells and that CPZ-induced cholestasis depends on the generation of oxidative stress. They also show that the certain susceptibility factors may be investigated.

Sodium/proton exchangers are ubiquitous secondary active transporters that can be found in all kingdoms of life. These proteins facilitate the transport of protons in exchange for sodium ions to help regulate internal pH, sodium levels, and cell volume. Na+/H+ exchangers belong to the SLC9 family and are involved in many physiological processes including cell proliferation, cell migration and vesicle trafficking. Dysfunction of these proteins has been linked to physiological disorders, such as hypertension, heart failure, epilepsy and diabetes. The goal of my thesis is to establish the molecular basis of ion exchange in Na+/H+ exchangers. By establishing how they bind and catalyse the movement of ions across the membrane, we hope we can better understand their role in human physiology. In my thesis, I will first present an overview of Na+/H+ exchangers and their molecular mechanism of ion translocation as was currently understood by structural and functional studies when I started my PhD studies. I will outline our important contributions to this field, which were to (i) obtain the first atomic structures of the same Na+/H+ exchanger (NapA) in two major alternating conformations, (ii) show how a transmembrane embedded lysine residue is essential for carrying out electrogenic transport, and (iii) isolate and recorde the first kinetic data of a mammalian Na+/H+ exchanger (NHA2) in an isolated liposome reconstitution system.

Current in vitro cellular methods utilised in drug metabolism and pharmacokinetic (DMPK) studies during drug development do not provide the 3D structure and functions of organs found in vivo, such that resulting in vitro-in vivo extrapolation (IVIVE) may not always accurately reflect clinical outcome. This highlights the need for the development of new dynamic in vitro cell models to aid improvement of IVIVE. The aim of this project was to incorporate characterised primary renal cells within a hollow fibre bioreactor for use in DMPK studies investigating renal clearance. Fluorescence based assays were developed to assess the functionality of three drug transporters involved in the renal transport of pharmaceutical compounds: P-gp, BCRP and OCT2. The developed assays were then applied alongside transporter visualisation and genetic expression assays to characterise primary human proximal tubule cells over a series of population doublings. Cells at a population doubling of 5 demonstrated the best transporter activity whilst allowing cells to be expanded in vitro. Polysulfone (PSF) based membranes, which are widely used in dialysis components were developed by blending additives to improve renal cell attachment and culture. The membranes exhibited a characteristic porous internal structure with smooth skin layers on the surface, and were able to be sterilised via autoclaving due to their high thermal stability. PSF blended with polyvinylpyrrolidone (PVP) was the most hydrophilic with cell metabolic activity similar to standard tissue culture plastic. The production of hollow fibres of varying thicknesses and properties from the PSF and PVP blend yielded a marked difference in renal cell attachment and long term viability. Fibres incorporated into glass casings to produce the single hollow fibre bioreactors (HFBs) were able to be sterilised by autoclaving whilst remaining intact. Due to the variation of fibre integrity within the batch, many fibres exhibited tears within the HFBs. This ultimately led to cell depletion within the fibre over the culture period; however, intact fibres demonstrated an increase in cell growth towards the end of the culture period under flow conditions. These results demonstrate the progress made towards a small scale in vitro renal model incorporating characterised primary renal cells to aid the improvement of IVIVE in DMPK research.

4 ABSTRACT Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Dimitrios Vagiannis Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Evaluation of antiproliferative effect of selected tyrosine kinase inhibitors in MDCKII cell lines Tyrosine kinases are important enzymes regulating crucial cellular processes including differentiation, proliferation, apoptosis, transcription, metabolism, and intercellular communication. Deregulation of these enzymes is the cause of various types of cancers. The blockade of their function by tyrosine kinase inhibitors (TKis) is considered a promising approach especially in antitumor pharmacotherapy. ATP-binding cassette (ABC) drug efflux transporters are a family of transmembrane proteins that pump a variety of structurally unrelated compounds out of the cell in an energy-dependent manner. They play an important role in pharmacokinetics (affect absorption, distribution, elimination) and, at the same time, can negatively influence efficacy of chemotherapy (participate in multidrug resistance phenomenon). In our research, we evaluated antiproliferative properties of four selected TKis, namely alectinib, brivanib, osimertinib and selumetinib, in MDCKII cell lines (parent one and those transduced with human...

L'homéostasie glutamatergique dans les fentes synaptiques régule la neurotransmission et préserve de l'excitotoxicité. Cela est particulièrement important dans l'oreille interne où il y a une libération soutenue de neurotransmetteur. Pour la plupart des cellules ciliées cochléaires et vestibulaires, la clairance du glutamate est assurée par les transporteurs du glutamate EAAT1 (GLAST) exprimés par les cellules de soutien. Un tel mécanisme n'est pas possible pour les cellules ciliées vestibulaires de type I car leur terminaison synaptique en calice empêche tout accès à la fente synaptique. Nous avons donc postulé qu'un ou plusieurs transporteurs du glutamate devaient être présents au niveau des cellules ciliées de type I ou du calice ou des deux.Grâce à des enregistrements électrophysiologiques, nous avons démontré qu'un courant anionique induit par le glutamate et bloqué par le DL-TBOA est présent dans les cellules ciliées de type I. Les techniques d'hybridation in situ et d'immunohistochimie ont révélé la présence d'EAAT4 et EAAT5. Ces deux transporteurs du glutamate, qui pourraient êtres à l'origine des courants enregistrés, sont exprimés par les cellules ciliées de type I et de type II. De plus, des expériences de RT-PCR et de microscopie électronique ont confirmé ces résultats et suggéré que ces transporteurs pourraient aussi être exprimés postsynaptiquement par le calice. Ces travaux de thèse montrent qu'EAAT4 et EAAT5, considérés respectivement comme spécifiques des tissus cérébelleux et rétiniens, ont une distribution plus large. Ces résultats posent la question des rôles potentiels de ces transporteurs dans l'homéostasie glutamatergique vestibulaire. / Glutamate homeostasis in synaptic clefts shape neurotransmission and prevent excitotoxicity. This may be particularly important in the inner ear where there is a continually high rate of neurotransmitter release. In the case of most cochlear and vestibular hair cells, clearance involves the diffusion of glutamate to supporting cells, where it is taken up by EAAT1 (GLAST), a glial glutamate transporter. A similar mechanism is unlikely to work in vestibular type I hair cells because the presence of calyx endings separates supporting cells from the synaptic zone. Based on this arrangement, we postulated that a glutamate transporter must be present in the type I hair cell, the calyx ending, or both. Using whole-cell patch-clamp recordings, we demonstrated that a glutamate-activated anion current blocked by DL-TBOA is expressed in type I hair cells. In situ hybridization and immunohistochemistry revealed that EAAT4 and EAAT5, two glutamate transporters that could support the anion current, are expressed in both type I and type II hair cells. Furthermore, RT-PCR and immunogold investigations confirmed those results and added that although preferentially expressed presynaptically, the transporters may also be present in the postsynaptic calyx membrane. Previously thought to be exclusively expressed in the cerebellum and retina respectively, this thesis work shows that EAAT4 and EAAT5 have a wider distribution. The potential role of these transporters in the glutamatergic homeostasis of the calyx synapse is then discussed.

La présence croissante de nanoparticules (NPs) dans les produits de la vie quotidienne (alimentation, médicaments, cosmétiques, textiles…) soulève de sérieuses inquiétudes quant à leurs potentiels effets nocifs pour la santé humaine. Les NPs de dioxyde de titane (TiO2) sont produites à l’échelle industrielle et peuvent déjà être trouvées dans plusieurs produits commerciaux tels que les peintures, les cosmétiques ou dans les systèmes de décontamination de l’eau ou de l’air. Dans le passé, les NPs de TiO2 étaient considérées comme inertes, mais, très récemment, l'Agence Internationale pour la Recherche sur le Cancer les a classées comme possiblement cancérogènes (groupe 2B) pour l’homme. De nombreuses études in vitro et in vivo ont démontré la potentielle neuro-toxicité des NPs de TiO2, mais très peu d'études se sont concentrées plus spécifiquement sur la barrière hémato-encéphalique (BHE), protégeant le cerveau. Aujourd'hui, en dépit des avancées constatées, la bio-cinétique et la bio-accumulation des NPs de TiO2 ainsi que les conséquences sur la physiologie de la barrière hémato-encéphalique (BHE) in vivo restent très peu documentées. De plus, dans l’évaluation du risque lié à l’exposition aux NPs, des facteurs de risque tel que l’âge ont jusqu’ici été quasiment ignorés. Dans ce contexte, l’objectif de ce projet est donc d’évaluer chez le rat adulte et âgé, l’impact d’une exposition aux NPs de TiO2 sur les fonctions de la BHE et sur le métabolisme cérébral. Nos résultats ont montré que les NPs de TiO2 s’accumulent dans certains organes et tissus (principalement dans les poumons, la rate et le foie) et ne sont pas distribuées au système nerveux central (SNC) que ce soit après injection intra-veineuse (IV) ou après une inhalation subaiguë à un nano-aérosol de TiO2. Après administration IV, une interaction directe entre NPs et les cellules endothéliales microvasculaires conduit à des altérations fonctionnelles au niveau de la BHE. Malgré l'absence de translocation vers le SNC, la bio-persistance du titane dans les organes périphériques semble être la cause de modulations de perméabilité de la BHE et d’une inflammation cérébrale. L'implication de médiateurs circulants faisant le lien entre la bio-persitance de titane dans les organes périphériques et les modulations observées au niveau cérébral a été démontré en utilisant un modèle in vitro de BHE. Une réponse exacerbée en termes de neuro-inflammation et de modulation de perméabilité de la BHE établit la vulnérabilité du cerveau âgé à la toxicité des NPs inhalées. Ces résultats ont démontré que malgré l'absence de translocation cérébrale, l'exposition aux NPs de TiO2 induit des altérations fonctionnelles de la BHE et une neuro-inflammation qui pourraient conduire à des troubles neurologiques. L’identification des médiateurs et la description des effets neurotoxiques restent encore à préciser. / The overwhelming presence of nanoparticles (NPs) in products including foods, medications, cosmetics, or textiles raises serious concerns about their potential harmful effects on human health. In the wide diversity of NPs, titanium dioxide (TiO2) NPs are among those produced on a large industrial scale and can already be found in several commercial products such as paints, cosmetics or in environmental decontamination systems. In the past, TiO2 NPs was considered inert, but, very recently, the International Agency for Research in Cancer (IARC) has classified TiO2 as possibly carcinogenic (group 2B) to human beings. Numerous in vitro and in vivo studies have shown the potential neuro-toxicity of TiO2 NPs, but very few studies focus on the central nervous system (CNS), Nowadays, notwithstanding the reported advances, the biokinetic and bioaccumulation ofTiO2 NPs and the consequences on the physiology of the blood-brain barrier (BBB) in vivo are unknown. In addition, NPs effect on susceptible population such as the elderly have been mostly ignored. In this context, the target of the present studies is to evaluate the in vivo impact of exposure to NPs on the BBB physiology and brain inflammation which could promote neurotoxicity in young adults and aging. Our results have shown that TiO2 NPs bioaccumulate in organs and tissues (lungs, spleen and liver especially) and don’t translocate to the brain either after IV or subacute inhalation exposure. In IV administration case, the direct interaction between NPs and brain endothelial cells induces BBB functional alterations. Despite the lack of CNS translocation, the biopersistence of titanium in peripheral organs may be indirectly the cause of BBB permeability alteration and brain inflammation. The involvement of circulating mediators linking titanium biopersitence in peripheral organs and brain impact has been demonstrated using an in vitro BBB model. An exacerbated response in term of neuro-inflammation and BBB permeability modulation has established the vulnerability of the aging brain to inhaled NPs toxicity. Taken together, our findings demonstrated that despite lack of brain translocation, exposure to TiO2 NPs induce BBB physiology alteration and neuro-inflammation that may lead to CNS disorders. Thereafter, identification of mediators and description of the neurotoxic effects may complete the assessment of the impact of TiO2 NPs exposure on the brain.