En transplantation d’organes, la réponse immunitaire de l’hôte contre son donneur reste une cause très importante de perte de greffons. Ainsi, un meilleur contrôle de la réponse par induction d’une tolérance spécifique reste une priorité en transplantation humaine. Dans ce projet,nous avons donc étudié l’homéostasie calcique au cours des phénomènes précoces de la maturation des cellules dendritiques (DC) humaines. Nous avons mis en évidence la présence d’une ECC (Entrée Capacitive de Calcium), dans la DC humaine, lors d’une stimulation par des agonistes des TLR (LPS ou Zymosan) ou des cytokines inflammatoires (TNF-α). De plus, cette ECC est gouvernée par le complexe ORAI-1 / STIM-1. En effet, l’absence d’expression de ces protéines induit une diminution de la maturation des DC humaines (phénotype, synthèse de cytokines). Par ailleurs, le Calcium, entré par l’ECC, va induire l’activation de canaux potassiques, sensibles à la concentration intracellulaire en Calcium : les canaux KCa3.1. Ces canaux, outre leur rôle dans la facilitation de l’ECC par hyperpolarisation de la membrane,contrôlent la migration des DC humaines. En effet, leur activation permet une sorte d’état de quiescence où les DC restent dans les tissus périphériques. L’ensemble de ces résultats obtenus,in vitro, suggèrent que ces canaux ioniques pourraient être une cible privilégiée pour le développement de nouvelles thérapies pour l’induction de tolérance immune en transplantation. / In organ transplantation, host immune response against donor still remains a major causeof graft loss. A better control of allogeneic response through the induction of specific tolerance isa major goal in human transplantation. In this work, we studied the calcium homeostasis during the earlier events of the human dendritic cells (DC) maturation. We showed the presence of a CCE(Capacitative Calcium Entry), in the human DC, during the stimulation by TLR agonists (LPS andZymosan) or inflammatory cytokines (TNF-α). Moreover, this CCE was managed by the ORAI-1/ STIM-1 complexe. Indeed, an inhibition of these proteins expression induced a decrease in theDC maturation (phebnotype, cytokines production). Then, the Calcium, issued to CCE, could activate potassium channels, sensitive to intracellular Calcium concentration: KCa3.1 channels.These channels, excepted their role in the facilitation of CCE by membrane hyperpolarisation,control the capacity of human DC migration. Their activation induced a kind of steady state where the DC staied in peripheral tissues. These results taken together suggest that the ion channels seemto be a good target for the development of new therapies to in order to promote allograft tolerance.

Até o momento a literatura não determinou qual o melhor estadio de maturação (imaturo ou maduro) para que o oocito mantenha sua competência para o desenvolvimento reprodutivo após a criopreservação. O objetivo deste estudo foi determinar em qual estadio meiótico (imaturo -VG (vesícula germinativa) ou maduro- MII (metáfase II)) o oócito é menos susceptível ao dano na criopreservação, utilizando modelo experimental bovino. Foram utilizados ovários de vacas abatidas em matadouro, após a aspiração dos folículos, os oócitos imaturos (VG) foram selecionados para a maturação in vitro e vitrificação, e foram divididos em três grupos: 1) oócitos maturados in vitro e não submetidos à vitrificação (CONTROLE); 2) oócitos vitrificados imaturos (VG), descongelados e submetidos à maturação in vitro (CRIO-MIV); 3) oócitos maturados in vitro (MII), vitrificados e descongelados (MIV-CRIO). Os oócitos foram avaliados quanto a: a)maturação nuclear pela técnica de orceína acética; b) integridade da zona pelúcida (ZP) através de microscopia de polarização; c) viabilidade oocitária pela técnica de DEAD-LIVE; d) desenvolvimento embrionário (taxa de clivagem, produção e eclosão de blastocistos) através da fertilização in vitro (FIV) e ativação partenogenética (AT). Não houve diferença na capacidade de maturação nuclear entre os oócitos frescos e descongelados no grupo CRIO-MIV (p=0,23). Em relação à zona pelúcida a totalidade dos oócitos (100%) nos três grupos apresentou leitura de zona pelúcida positiva, não havendo correlação com evolução embrionária posterior. Na análise de viabilidade celular pelo DEAD-LIVE verificou-se que houve redução da viabilidade do grupo MIV-CRIO (27%) quando comparado com controle (84%) (p<0,0001). Na análise do potencial de desenvolvimento embrionário o grupo controle apresentou melhores taxas de clivagem após FIV (80%) e AT (58%), do que os grupos CRIO-MIV (28%; p<0,0001; 28%; p=0,0002, respectivamente) e MIV-CRIO (26%; p<0,0001; 22%, p<0,0001,respectivamente). As taxas de formação de blastocisto e eclosão após FIV nos grupos CRIO-MIV, MIV-CRIO e após AT no grupo MIV-CRIO foram nulas. Houve a produção e eclosão de apenas um blastocisto no grupo CRIO-MIV após AT. No modelo experimental utilizado, o procedimento de vitrificação comprometeu parcialmente a viabilidade dos oócitos medida pela técnica de DEAD- LIVE e completamente o desenvolvimento embrionário subseqüente, independente do estadio de maturação meiótica (VG ou MII) durante a criopreservação. No entanto, oócitos vitrificados em estadio de VG e submetidos à MIV foram meioticamente competentes e progrediram até o estadio de MII, sugerindo que o dano não compromete a capacidade de maturação nuclear do oócito. Este estudo não conseguiu determinar qual o melhor estadio meiótico oocitário para criopreservação, já que os dois estadios meióticos (VG e MII) se mostraram igualmente prejudicados pela criopreservação em relação à capacidade reprodutiva. / Until the present literature has not achieved a consensus regarding the best maturation stage for oocyte to maintain their reproductive capacity after cryopreservation. The aim of this study was to determine, using an experimental bovine model, in which stage of development (VG stage, immature, or MII stage, post-maturation in vitro) the oocyte is less susceptible to damage during cryopreservation. Immature oocytes (VG) from the ovaries of slaughtered cows were selected for in vitro maturation or vitrification and divided into three groups. The first group (CONTROL) consisted of immature oocytes, matured in vitro without vitrification; the second group (CRYO-IVM) consisted of vitrified immature oocytes thawed and submitted to in vitro maturation; and the third group (IVM-CRYO) consisted of matured in vitro oocytes submitted to vitrification and thawing. The oocytes were evaluated for: nuclear maturation by acetic orcein staining; integrity of the zona pellucida using a polarized microscope; cell viability by the Dead-Live technique; and embryo development (cleavage, production and hatching rate) by in vitro fertilization and parthenogenetic activation. There was no difference in capacity of nuclear maturation between fresh and thawed oocytes (p=0.23). Regarding the zona pellucida (ZP), all oocytes (100%) of all three groups (control, CRYO-IVM and IVMCRYO) presented a positive ZP reading, with no correlation with later embryo evolution. DEAD-LIVE analysis of cell viability revealed reduction of viability in the IVM-CRYO group (27%) compared to control (84%) (p<0.0001) and to the CRYO-IVM group (56%) (p=0.017), with no difference between the last two groups (p=0.055). Analysis of the potential for embryo development by means of in vitro fertilization showed that the control group presented better cleavage and blastocyst formation rates than the CRYO-IVM (p<0.0001 and p<0.0001, respectively) and the IVM-CRYO (p<0.0001 and p=0.0004, respectively) groups. Analyzing the potential for embryo development the control group presented better cleavage by means of in vitro fertilization (80%) and parthenogenetic activation (58%) than the CRYOIVM (28%; p<0,0001; 28%; p=0,0002, respectively) and the IVM-CRYO groups (26%; p<0,0001; 22%, p<0,0001,respectively) Analysis of blastocyst formation rates and hatching after FIV and AT in CRYO-IVM and IVM-CRYO groups were null. Vitrification of bovine oocytes causes great impairment of their reproductive capacity regardless of the stage of maturation at the time of freezing. However the vitrified immature oocytes submitted to IVM maintained their capacity of nuclear maturation, as they achieved MII stage. This study was not able to determine which stage was better in reducing crio damage, as both stages (VG and MII) presented equally impaired by the process.

L’objectif de ce travail était de comparer l’importance de la composante nerveuse dans les différences de production de force entre les enfants et les adultes en tenant compte des conditions mécaniques qui sont susceptibles de l’influencer (longueur musculaire, mode et vitesse de contraction) et des effets de la fatigue. Trente et un garçons pré-pubères de 8-12 ans et 37 hommes de 18-30 ans ont été recrutés et répartis dans les 3 études composant ce projet. La première étude portait sur les effets de la longueur musculaire et de la fatigue sur la production de force et le niveau d’activation maximal volontaire (VA) des extenseurs du genou.La seconde traitait des effets de la longueur musculaire sur la production de force et le VA des fléchisseurs plantaires. Enfin, la troisième portait sur les effets du mode et de la vitesse de contraction sur le VA des extenseurs du genou. Les propriétés neuromusculaires ont été évaluées à l’aide de stimulations magnétiques périphériques et de contractions maximales volontaires. Les résultats montrent un VA des extenseurs du genou supérieur chez l’adulte lors de grande longueur musculaire (90°-100° de flexion) (étude 1), mais aucune différence avec l’enfant à courte longueur musculaire (20° de flexion). En revanche, sur des groupes musculaires tels que les fléchisseurs plantaires, aucun effet de la longueur sur le VA n’a été observé chez les enfants et les adultes (étude 2). En ce qui concerne les effets du mode de contraction, nos résultats montrent que le VA est inférieur en conditions excentrique et concentrique par rapport à la condition isométrique, mais aucune différence n’était observée entre les groupes (étude 3). Toutefois, l’effet de cette baisse de VA sur la production de force variait entre les groupes ; une diminution de force accompagnant la diminution de VA n’était retrouvée que chez l’adulte. Enfin,nous avons montré que la baisse du VA au cours d’un protocole de fatigue est plus conséquente chez les enfants par rapport aux adultes (étude 1), ce qui témoigne de l’existence d’une fatigue centrale majorée chez l’enfant. Une maturation inaboutie et les propriétés musculo-tendineuses particulières de l’enfant (compliance supérieure) pourraient expliquer les résultats obtenus au cours de ce travail. / The purpose of this PhD thesis was to compare the contribution of the maximal voluntary activation level (VA) of the motor units to force production differences between children and adults in various mechanical conditions that affect force production (muscle length, contraction mode and velocity), and in fatigue condition. Thirty one pre-pubertal 8 to 12-year old boys and 37 men (18-30 years) were recruited and allocated into the 3 studies ofthis project. The first study was devoted to compare the effects of muscle length and fatigue on the VA and force generating capacity of the knee extensors between children and adults. In the second study, we compared the effects of muscle length on the VA and force generating capacity of the plantar flexors between children and adults. The third study analysed the effect of contraction mode and velocity on the VA and force generating capacity of the knee extensors between children and adults. Neuromuscular properties were assessed with peripheral magnetic stimulation and maximal voluntary contractions. Results showed a higher VA of the knee extensors in adults at long muscle length (90° and 100° knee flexion) but no difference between children and adults at short length (20° knee flexion; study 1). However, the VA of the plantar flexors was not affected by muscle length changes whatever the age group considered (study 2). Results also showed a higher VA in isometric mode compared to eccentric and concentric conditions whatever the age group (study 3). However, the effect of this VA reduction on force generating capacity differed between groups, a concomitant force reduction being observed in adults, but not in children. Finally, we observed a greater VA reduction and therefore greater central fatigue in children during the fatiguing protocol (study 1). A relative immaturity and the particular musculo-tendinous properties of children (higher compliance) may account for the reported results.

L’activité RNase P est l'activité essentielle qui élimine les séquences 5' supplémentaires des précurseurs d'ARN de transfert. "PRORP" (PROteinaceous RNase P) définit une nouvelle catégorie de RNase P uniquement protéique. Avant la caractérisation de PRORP, on pensait que les enzymes RNase P étaient universellement conservées sous forme de ribonucléoprotéines (RNP). La caractérisation de PRORP a révélé une enzyme avec deux domaines principaux, un domaine N-terminal contenant plusieurs motifs PPR et un domaine NYN C-terminal portant l’activité catalytique. Nous avons utilisé une combinaison d'approches biochimiques et biophysiques pour caractériser le complexe PRORP / ARNt. La structure du complexe en solution a été déterminée par diffusion des rayons X aux petits angles (SAXS) et les Kd des interactions de différents mutants de PRORP avec l’ARNt ont été déterminées par ultracentrifugation analytique. Notre analyse révèle un cas intéressant d'évolution convergente. Il suggère que PRORP a développé un processus de reconnaissance de l'ARN similaire à celui des RNase P RNP. Par ailleurs, nous avons mis en place une approche de co-immunoprécipitation de PRORP avec l’ARN afin de définir le spectre de substrats des RNase P protéiques. / RNase P is the essential activity that removes 5'-leader sequences from transfer RNA precursors. “PRORP” (PROteinaceous RNase P) defines a novel category of protein only RNase P. Before the characterization of PRORP, RNase P enzymes were thought to occur universally as ribonucleoproteins (RNP). The characterization of PRORP revealed an enzyme with two main domains, an N-terminal domain containing multiple PPR motifs and a C-terminal NYN domain holding catalytic activity. We used a combination of biochemical and biophysical approaches to characterize the PRORP / tRNA complex. The structure of the complex in solution was determined by small angle X-ray scattering and Kd values of the PRORP / tRNA interaction were determined by analytical ultracentrifugation. We also analyzed direct interaction of a collection of PPR mutants with tRNA in order to determine the relative importance of individual PPR motifs for RNA binding. This reveals to what extent PRORP target recognition process conforms to the mode of action of PPR proteins interacting with linear RNA. Altogether, our analysis reveals an interesting case of convergent evolution. It suggests that PRORP has evolved an RNA recognition process similar to that of RNP RNase P. Moreover, we also implemented a PRORP-RNA co-immunoprecipitation approach to determine the full extent of PRORP substrates.

L’imagerie médicale et la 3D, en pleine expansion dans le champ de l'anthropologie biologique, permettent d’explorer les structures internes tout en les préservant. L’étude de la micro-architecture osseuse trabéculaire permet d’appréhender la variabilité de l'os humain à une échelle jusqu'à présent peu explorée. Dans le cadre de cette recherche, cette variabilité est analysée et caractérisée en termes de croissance et de maturation, en fonction des critères individuels d’âge et de sexe, ainsi que dans des contextes pathologiques variés. Les images microtomodensitométriques des métaphyses humérales proximales de 43 sujets immatures (provenant de 3 collections ostéologiques de référence et couvrant l’ensemble des âges du développement) et celles de 8 cas paléopathologiques (représentant 5 étiologies différentes) ont été analysées pour quantifier la micro-architecture osseuse trabéculaire. Nos résultats montrent que cette micro-architecture varie pendant et entre les différentes phases de la croissance. Des corrélations avec l’âge sont mises en évidence, si elles n’expliquent pas suffisamment la variabilité observée pour en faire des estimateurs d'âge précis, il apparaît néanmoins que les variations relevées entre les différents volumes d’intérêt pourraient caractériser différentes périodes de la croissance. Les variables mesurées présentent des différences sexuelles significatives pendant l’adolescence, mais ne peuvent pas en l'état être utilisées pour la diagnose sexuelle. L'étude de la microarchitecture trabéculaire osseuse des sujets pathologiques atteste d’un développement anormal de l’os et donc du statut pathologique de l’individu observé. / Medical imaging and 3D reconstructions are used increasingly by anthropologists; they allow both investigating and preserving internal structures. Study of trabecular bone microarchitecture allows understanding variability of human skeleton at a smaller scale. This variability is observed and characterized in terms of normal growth and maturation according to age and sex, and for several pathological conditions. μCT scans of proximal metaphysis of humerus from 43 immature individuals (coming from 3 identified skeletons collections and representing all periods of age development) and 8 paleopathological cases (corresponding to 5 different etiologies) have been analyzed to quantify bone microarchitecture. Our results show that this microarchitecture varies during and between different phases of growth. Correlations with age are highlighted, even if they do not sufficiently explain the observed variability in order to represent specific age estimators; it nevertheless appears that the variations observed between the different volumes of interest could characterize different periods of growth. The measured variables showed significant sex differences only during the adolescence period, but they cannot be used, in the present state, for sex determination. The study of the trabecular bone microarchitecture of pathological individuals attests of the abnormal development of bone and therefore of their pathological status.

Résumé La Ribonucléase P (RNase P) est une enzyme principalement reconnue pour sa participation à la maturation en 5’des ARN de transfert (ARNt). Cependant, d’autres substrats sont reconnus par l’enzyme. En général, la RNase P est composée d’une sous-unité ARN (le P-ARN, codé par le gène rnpB) qui porte le centre actif de l’enzyme et d’une ou de plusieurs sous-unités protéiques (la P-protéine). Les P-ARN chez toutes les bactéries, la majorité des archéobactéries et dans le génome nucléaire de la plupart des eucaryotes, possèdent généralement une structure secondaire très conservée qui inclut le noyau (P1-P4); l’hélice P4 constitue le site catalytique de l’enzyme et l’hélice P1 apparie les extrémités du P-ARN en stabilisant sa structure globale. Les P-ARN mitochondriaux sont souvent moins conservés et difficiles à découvrir. Dans certains cas, les seules régions de structure primaire qui restent conservées sont celles qui définissent le P4 et le P1. Pour la détection des gènes rnpB, un outil de recherche bioinformatique, basé sur la séquence et le profil de structure secondaire, a été développé dans le laboratoire. Cet outil permet le dépistage de toutes les séquences eucaryotes (nucléaires et mitochondriales) du gène avec une très grande confiance (basée sur une valeur statistique, E-value). Chez les champignons, plusieurs ascomycètes encodent un gène rnpB dans leur génome mitochondrial y compris tous les membres du genre d’Aspergillus. Cependant, chez les espèces voisines, Neurospora crassa, Podospora anserina et Sordaria macrospora, une version mitochondriale de ce gène n’existe pas. Au lieu de cela, elles contiennent deux copies nucléaires du gène, légèrement différentes en taille et en contenu nucléotidique. Mon projet a été établi dans le but d’éclaircir l’évolution de la RNase P mitochondriale (mtRNase P) chez ces trois espèces voisines d’Aspergillus. En ce qui concerne les résultats, des modèles de structures secondaires pour les transcrits de ces gènes ont été construits en se basant sur la structure consensus universelle de la sous-unité ARN de la RNase P. Pour les trois espèces, par la comparaison de ces modèles, nous avons établi que les deux copies nucléaires du gène rnpB sont assez distinctes en séquence et en structure pour pouvoir y penser à une spécialisation de fonction de la RNase P. Chez N. crassa, les deux P-ARN sont modifiés probablement par une coiffe et les extrémités 5’, 3’ sont conformes à nos modèles, ayant un P1 allongé. Encore chez N. crassa, nous avons constaté que les deux copies sont transcrites au même niveau dans le cytoplasme et que la plus petite et la plus stable d’entre elles (Nc1) se retrouve dans l’extrait matriciel mitochondrial. Lors du suivi du P-ARN dans diverses sous-fractions provenant de la matrice mitochondriale soluble, Nc1 est associée avec l’activité de la RNase P. La caractérisation du complexe protéique, isolé à partir de la fraction active sur un gel non dénaturant, révèle qu’il contient au moins 87 protéines, 73 d’entre elles ayant déjà une localisation mitochondriale connue. Comme chez la levure, les protéines de ce complexe sont impliquées dans plusieurs fonctions cellulaires comme le processing de l’ADN/ARN, le métabolisme, dans la traduction et d’autres (par exemple : la protéolyse et le repliement des protéines, ainsi que la maintenance du génome mitochondrial). Pour trois protéines, leur fonction est non déterminée. / Abstract Ribonuclease P (RNase P) is an endonuclease that cleaves 5’- leader sequences from tRNA precursors and a few other small RNAs. In most cases, the enzyme is a ribonucleo-protein complex (ribozyme), containing an RNA subunit (P-RNA; encoded by the rnpB gene) that carries the active centre of the enzyme, plus one or more protein subunits. P-RNAs in Bacteria, Eukarya and Archaea have a highly conserved secondary structure including the core P1 and P4 helices. P4 forms the catalytic site of the ribozyme, and P1 pairs the RNA termini, stabilizing overall structure and protecting from nuclease degradation. For processing of mitochondrial (mt) tRNAs, certain eukaryotic species (e.g., Saccharomyces cerevisiae, Aspergillus nidulans) have separate mtDNA-encoded P-RNAs (of bacterial origin). Mt P-RNAs are often less conserved, and difficult to discover. To identify rnpB genes, we have developed a search tool based on sequence plus secondary structure profiles. It predicts all known eukaryotic (nuclear and organellar) rnpB genes with high confidence (based on E-values). In fungi, many ascomycetes encode a mitochondrial rnpB gene, including all members of Aspergillus. Yet, the closely related Neurospora crassa, Podospora anserina and Sordaria macrospora lack an mtDNA-encoded gene version. Instead, they contain two nuclear gene copies with slightly different sequences. My project aims to elucidate the evolution of mitochondrial RNase P in these three closely related species. We have established secondary structure models based on comparisons with the universal minimum consensus secondary structure for all nuclear gene mtP-RNAs copies in all three species. By comparison of these secondary structure models, we have established that the two nuclear copies of rnpB gene are quite distinct in sequence and structure, suggesting a specialization of function. In N. crassa, both P-RNAs are modified most likely by capping, and 5’- 3’ termini perfectly conform to P-RNA structure models that have an elongated P1 helical pairing. Furthermore, we find that the two nuclear copies of rnpB gene are present at about the same level in the cytoplasm, and that the shorter form of P-RNA (Nc1) translocates into the (soluble) mitochondrial matrix. When tracing P-RNA in different mitochondrial sub-fractions of a native gel, the presence of Nc1 and mitochondrial RNase P activity are associated. A proteomics characterization of a P-RNA complex isolated by native gel electrophoresis reveals that it contains at least 87 proteins, 73 of which are of known mitochondrial localization. Like in yeast, the complex contains proteins potentially involved in other DNA/RNA processing activities, but also in translation, in metabolism, and in protein folding. Only three proteins are of unknown function.

Les anticorps thérapeutiques sont des molécules de choix pour le traitement standard de nombreuses formes de cancers. Leur application est à ce jour restreinte au compartiment extracellulaire à cause de leur taille trop importante qui les empêche de traverser la membrane cellulaire. Comme la plupart des cibles thérapeutiques du cancer semblent être situées dans le milieu intracellulaire, ce serait un plus de pouvoir exploiter les propriétés des anticorps dans les cellules pour étudier et perturber l’activité de ces cibles. Néanmoins, l’utilisation des anticorps dans le milieu intracellulaire constitue un véritable challenge, notamment à cause de la membrane cellulaire et de l’environnement réducteur du cytoplasme. L’ensemble des travaux de thèse présentés dans ce manuscrit ont permis d’établir les bases de plusieurs stratégies innovantes d’immunociblage intracellulaire et de mettre en lumière l’importance des différentes étapes de validation d’anticorps ou de fragments dérivés utilisés comme anticorps intracellulaires. La vectorisation d’anticorps complets par électroporation, le développement d’un intracorps bispécifique original anti-PCNA et la mise au point d’une méthode de mutagenèse inspirée de l’hypermutation somatique constituent les principales avancées apportées par ce travail dans le domaine de la recherche technologique en immuno biotechnologie. / Therapeutic antibodies are interesting molecules used to treat numerous pathologies such as cancer. Because of their size, their application is currently limited to the extracellular space. Indeed, antibodies cannot cross the cell membrane. Almost all therapeutic targets in cancer seem to be located inside cells, it would be beneficial to take advantage of antibodies in cells in order to neutralize the activity of these targets. The use of antibodies inside the cells is a real challenge, because of the cell membrane and the reducing environment of the cytoplasm. Several strategies of intracellular immunotargeting are presented in this thesis.

Chez les procaryotes, les systèmes ABC (ATP-binding cassette) canoniques permettent un import efficace et de haute affinité du nickel, en grande partie via l'action de la Ni-BP (nickel-binding protein) qui va jouer le rôle de récepteur extracytoplasmique du nickel avant son passage à travers la membrane interne. Dans ces travaux, nous avons cherché à mieux caractériser les stratégies d'import de nickel par les bactéries, notamment par l'étude cristallographique des Ni-BP. La résolution des structures de cinq de ces protéines, en interaction avec du nickel, nous a permis d'identifier les sites de fixation et ainsi de mettre en évidence les différents modes d'interaction de ce métal avec les Ni-BP. Nos résultats montrent notamment que la coordination du nickel requiert toujours la présence d'un métallophore exogène, appelé nickelophore. En parallèle, des études in vivo ont été conduites sur Escherichia coli, montrant que cette bactérie semble être capable de synthétiser son propre nickelophore. D'autre part, ces travaux ont permis la résolution de la structure de HypB de Helicobacter pylori en interaction avec du nickel. Celle-ci permet de mieux appréhender le rôle central de cette protéine au sein des voies de maturation des enzymes à nickel. / In prokaryotes, canonical ABC (ATP-binding cassette) importers allow an efficient uptake of nickel with high affinity through the inner membrane, largely via the action of the extracytoplasmic nickel-binding protein (Ni-BP). In this work, we intended to better understand the strategies developed by bacteria to scavenge nickel in the environment, especially through the structural studies of several Ni-BP. The resolution of the crystal structures of five Ni-BPs from diverse bacteria, in interaction with nickel, led us to identify their nickel-binding sites and shed light on the different binding modes. Our results show that the presence of an exogenous metallophore, called nickelophore, is always required. In vivo studies in Escherichia coli were also conducted, showing that this bacteria seems to be able to synthesize its own nickelophore. In addition, we have solved the crystal structure of Helicobacter pylori HypB in complex with nickel. This result provides insight into its cellular function in nickel enzyme maturation pathways.

Made available in DSpace on 2016-12-08T16:24:20Z (GMT). No. of bitstreams: 1 PGCA15MA161.pdf: 119035 bytes, checksum: a5dfaf04161545272882e1423c258dc0 (MD5) Previous issue date: 2015-02-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The in vitro embryo production has a strong commercial and preservationist impact in Brazil. However, there are still barriers to be overcome, since the in vitro maturation does not mimic the in vivo maturation. The maturation optimization increases the embryo competence and quality, making them more cryotolerants. The meiotic blockage, after oocytes removal from their follicular environment, may be a good alternative. Three experiments (n = 2848) evaluated the Cordicepim as a meiotic blocker in bovine oocytes in different culture media, and its influence on embryonic development. Follicles 3-8 mm diameter, were punctured from abattoir ovaries and allocated into 4 groups: Control (TCM-199 + pyruvate, FSH, LH and SFB); MIVCord (TCM-199 + Pyruvate, FH, LH, FCS, Cordicepim); TCMCor (TCM-199 + Cordicepim) and TCMCont (TCM-199), followed by standard maturation (IVM) for 20 or 24 h. The effect of meiosis blockage in embryo production was assessed by cleavage and blastocyst rate, and total number of cells assessed. To evaluate the stage of maturation, oocytes were stained with Hoechst and evaluated by microscopy. Quantitative data were analyzed by ANOVA and Tukey test and the qualitative data by chi-square, with 5% significance. Cordicepim did not affect cleavage (60.7 vs. 56.4%) and blastocysts (30.7 vs. 24.8%) rates when added to supplemented medium (MIVCord). However, there was a reduction in cleavage (42%) and blastocysts (12.1%) rates when added to a medium without supplements (TCMCord). On the kinetics of maturation after 6 hours of pre culture, only TCMCord group maintained most oocytes blocked. Cell density of the produced embryos (26 h maturation) in MIVcont groups (189.2 ± 11.4), MIVCord (187.1 ± 12.1) and TCMCont (171.2 ± 10.4) did not differ and were higher than TCMCord group (119.7 ± 11.4). Already, with 30 h maturation cell density was similar between embryos from control group (224.2 ± 17.5) and TCMCord group (240.1 ± 10.4). However, even with 30 h maturation, only 64.8% of the oocytes completed maturation in TCMCord group, while 100% maturated in Control group. We conclude that 6 h of culture in cordicepim in the absence of gonadotropins, partially inhibits the meiosis in oocytes, and this inhibition is not successfully reversed even after 24 hours of maturation pattern in the absence of cordicepim. The cordicepim reduces embryo production when in vitro maturation is performed for 26 h. However, within 30 h maturation time, the Cordicepim does not affect embryo production rate. Yet, the cordicepim prevents the cumulus cells expansion in an irreversible manner / A produção in vitro de embriões tem um forte impacto comercial e preservacionista no Brasil. Entretanto ainda existem barreiras a serem superadas, já que a maturação in vivo não é totalmente mimetizada pela maturação in vitro. A otimização da maturação pode aumentar a competência e a qualidade embrionária, tornando-os mais criotolerantes. Uma boa alternativa pode ser o bloqueio meiótico reversível. Três experimentos (n = 2848) avaliaram o Cordicepim como bloqueador meiótico para oócitos bovinos, em diferentes meios de cultivo e sua influência no desenvolvimento embrionário. Folículos de 3-8 mm, obtidos por aspiração de ovários de frigorífico foram distribuídos em 4 grupos: Controle (TCM-199 + piruvato, FSH, LH e SFB) MIVCord (TCM-199 +Piruvato, FH, LH, SFB, Cordicepim) TCMCor (TCM-199 + Cordicepim) e TCMCont (TCM-199), seguido de maturação padrão (MIV) por 20 ou 24 horas. O efeito do bloqueio meiótico na produção de embriões foi avaliado pela taxa de clivagem, blastocisto e número total de células. Para avaliação do estágio de maturação, os oócitos foram corados com Hoechst e avaliados por microscopia. Os dados quantitativos foram submetidos à ANOVA e teste de Tukey e os qualitativos ao qui-quadrado, com 5% de significância. O Cordicepim não influenciou as taxas de clivagem (60,7 vs 56,4%) e blastocistos (30,7 vs 24,8%), quando empregado em meio suplementado (MIVCord). Porém, houve uma redução na taxa de clivagem (42%) e blastocistos (12,1%), quando empregado em meio sem suplementos (TCMCord). Na cinética de maturação nuclear após 6 horas de pré-maturação, apenas o grupo TCMCord manteve a maioria dos oócitos bloqueados (67%). A densidade celular dos embriões produzidos (26 h maturação) nos grupos controle (189,2 ± 11,4), MIVCord (187,1 ± 12,1) e TCMCont (171,2 ± 10,4) não diferiram entre si e foram superiores ao grupo TCMCord (119,7 ± 11,4). Já com 30 h de maturação a densidade celular dos embriões foi semelhante entre os grupos controle 30 (224,2 ± 17,5) e TCMCord 30 (240,1 ± 10,4). Porém, mesmo com 24 h de maturação padrão, apenas 64,8% dos oócitos do grupo TCMCord 30 completaram a maturação, enquanto no grupo controle 100% maturaram. Conclui-se que 6 h de pré-maturação em cordicepim, na ausência de suplementos, inibe parcialmente a meiose em oócitos bovinos, porém esta inibição não é revertida com êxito mesmo após 24 horas de maturação padrão na ausência de cordicepim. O cordicepim reduz a produção de embriões in vitro com 26 h de maturação. Já com 30 h de maturação, o Cordicepim não influencia a taxa de produção embrionária. Ainda, o cordicepim impede de forma irreversível a expansão das células do cumulus

Até o momento a literatura não determinou qual o melhor estadio de maturação (imaturo ou maduro) para que o oocito mantenha sua competência para o desenvolvimento reprodutivo após a criopreservação. O objetivo deste estudo foi determinar em qual estadio meiótico (imaturo -VG (vesícula germinativa) ou maduro- MII (metáfase II)) o oócito é menos susceptível ao dano na criopreservação, utilizando modelo experimental bovino. Foram utilizados ovários de vacas abatidas em matadouro, após a aspiração dos folículos, os oócitos imaturos (VG) foram selecionados para a maturação in vitro e vitrificação, e foram divididos em três grupos: 1) oócitos maturados in vitro e não submetidos à vitrificação (CONTROLE); 2) oócitos vitrificados imaturos (VG), descongelados e submetidos à maturação in vitro (CRIO-MIV); 3) oócitos maturados in vitro (MII), vitrificados e descongelados (MIV-CRIO). Os oócitos foram avaliados quanto a: a)maturação nuclear pela técnica de orceína acética; b) integridade da zona pelúcida (ZP) através de microscopia de polarização; c) viabilidade oocitária pela técnica de DEAD-LIVE; d) desenvolvimento embrionário (taxa de clivagem, produção e eclosão de blastocistos) através da fertilização in vitro (FIV) e ativação partenogenética (AT). Não houve diferença na capacidade de maturação nuclear entre os oócitos frescos e descongelados no grupo CRIO-MIV (p=0,23). Em relação à zona pelúcida a totalidade dos oócitos (100%) nos três grupos apresentou leitura de zona pelúcida positiva, não havendo correlação com evolução embrionária posterior. Na análise de viabilidade celular pelo DEAD-LIVE verificou-se que houve redução da viabilidade do grupo MIV-CRIO (27%) quando comparado com controle (84%) (p<0,0001). Na análise do potencial de desenvolvimento embrionário o grupo controle apresentou melhores taxas de clivagem após FIV (80%) e AT (58%), do que os grupos CRIO-MIV (28%; p<0,0001; 28%; p=0,0002, respectivamente) e MIV-CRIO (26%; p<0,0001; 22%, p<0,0001,respectivamente). As taxas de formação de blastocisto e eclosão após FIV nos grupos CRIO-MIV, MIV-CRIO e após AT no grupo MIV-CRIO foram nulas. Houve a produção e eclosão de apenas um blastocisto no grupo CRIO-MIV após AT. No modelo experimental utilizado, o procedimento de vitrificação comprometeu parcialmente a viabilidade dos oócitos medida pela técnica de DEAD- LIVE e completamente o desenvolvimento embrionário subseqüente, independente do estadio de maturação meiótica (VG ou MII) durante a criopreservação. No entanto, oócitos vitrificados em estadio de VG e submetidos à MIV foram meioticamente competentes e progrediram até o estadio de MII, sugerindo que o dano não compromete a capacidade de maturação nuclear do oócito. Este estudo não conseguiu determinar qual o melhor estadio meiótico oocitário para criopreservação, já que os dois estadios meióticos (VG e MII) se mostraram igualmente prejudicados pela criopreservação em relação à capacidade reprodutiva. / Until the present literature has not achieved a consensus regarding the best maturation stage for oocyte to maintain their reproductive capacity after cryopreservation. The aim of this study was to determine, using an experimental bovine model, in which stage of development (VG stage, immature, or MII stage, post-maturation in vitro) the oocyte is less susceptible to damage during cryopreservation. Immature oocytes (VG) from the ovaries of slaughtered cows were selected for in vitro maturation or vitrification and divided into three groups. The first group (CONTROL) consisted of immature oocytes, matured in vitro without vitrification; the second group (CRYO-IVM) consisted of vitrified immature oocytes thawed and submitted to in vitro maturation; and the third group (IVM-CRYO) consisted of matured in vitro oocytes submitted to vitrification and thawing. The oocytes were evaluated for: nuclear maturation by acetic orcein staining; integrity of the zona pellucida using a polarized microscope; cell viability by the Dead-Live technique; and embryo development (cleavage, production and hatching rate) by in vitro fertilization and parthenogenetic activation. There was no difference in capacity of nuclear maturation between fresh and thawed oocytes (p=0.23). Regarding the zona pellucida (ZP), all oocytes (100%) of all three groups (control, CRYO-IVM and IVMCRYO) presented a positive ZP reading, with no correlation with later embryo evolution. DEAD-LIVE analysis of cell viability revealed reduction of viability in the IVM-CRYO group (27%) compared to control (84%) (p<0.0001) and to the CRYO-IVM group (56%) (p=0.017), with no difference between the last two groups (p=0.055). Analysis of the potential for embryo development by means of in vitro fertilization showed that the control group presented better cleavage and blastocyst formation rates than the CRYO-IVM (p<0.0001 and p<0.0001, respectively) and the IVM-CRYO (p<0.0001 and p=0.0004, respectively) groups. Analyzing the potential for embryo development the control group presented better cleavage by means of in vitro fertilization (80%) and parthenogenetic activation (58%) than the CRYOIVM (28%; p<0,0001; 28%; p=0,0002, respectively) and the IVM-CRYO groups (26%; p<0,0001; 22%, p<0,0001,respectively) Analysis of blastocyst formation rates and hatching after FIV and AT in CRYO-IVM and IVM-CRYO groups were null. Vitrification of bovine oocytes causes great impairment of their reproductive capacity regardless of the stage of maturation at the time of freezing. However the vitrified immature oocytes submitted to IVM maintained their capacity of nuclear maturation, as they achieved MII stage. This study was not able to determine which stage was better in reducing crio damage, as both stages (VG and MII) presented equally impaired by the process.