Secretoneurin (SN) is a 31-43 amino acid, functional peptide derived by proteolytic processing from the middle domain of the ~600 amino acid secretogranin-II (SgII) precursor. In teleosts there are 2 forms arising from 2 different genes, SgIIa and SgIIb. In turn, there are both SNa and SNb in teleost. Secretoneurin is a well-conserved peptide during evolution from fish to mammals and widely distributed in secretory granules of endocrine cells and neurons. Secretoneurin plays important roles in different biological processes, for example controlling vertebrate reproduction by stimulating luteinizing hormone release from the pituitary. A potential new role of SN in feeding in goldfish is the subject of the research presented in this thesis. Firstly, we looked at the distribution of SgIIa mRNA in various female goldfish tissues using both RT-PCR and Q-PCR techniques in order to determine which tissue expresses SgIIa mRNA and in which level. We found that SgIIa mRNA was detected in different amounts in all tissues examined. The main tissues of interest were hypothalamus, telencephalon and gut, they all expressed SgIIa. Secondly, we examined the effect of acute (26 h), short (3 days), medium (7 days) and long (14 days) fasting and periprandial changes on SgIIa mRNA level in hypothalamus, telencephalon and gut using Q-PCR method. The results showed that SgIIa mRNA increases under the effect of acute and short fasting, however, medium and long fasting did not affect SgIIa mRNA. Thirdly, we examined the effect of brain injection of goldfish SNa on food intake and locomotor behavior and the expression of some feeding neuropeptides such as neuropeptide Y, orexin, chocystokinin and cocaine-and amphetamine-regulated transcript I after treatment. Injection of SNa in the third brain ventricle increased food intake and fish activity. Associated with this was an increase in NPY and decrease in CARTI mRNA levels in hypothalamus. The increase in SgIIa mRNA following fasting and the increase of food intake as a result of SNa treatment suggest a novel role for SNa in feeding processes.

Secretoneurin (SN) is a 31-43 amino acid, functional peptide derived by proteolytic processing from the middle domain of the ~600 amino acid secretogranin-II (SgII) precursor. In teleosts there are 2 forms arising from 2 different genes, SgIIa and SgIIb. In turn, there are both SNa and SNb in teleost. Secretoneurin is a well-conserved peptide during evolution from fish to mammals and widely distributed in secretory granules of endocrine cells and neurons. Secretoneurin plays important roles in different biological processes, for example controlling vertebrate reproduction by stimulating luteinizing hormone release from the pituitary. A potential new role of SN in feeding in goldfish is the subject of the research presented in this thesis. Firstly, we looked at the distribution of SgIIa mRNA in various female goldfish tissues using both RT-PCR and Q-PCR techniques in order to determine which tissue expresses SgIIa mRNA and in which level. We found that SgIIa mRNA was detected in different amounts in all tissues examined. The main tissues of interest were hypothalamus, telencephalon and gut, they all expressed SgIIa. Secondly, we examined the effect of acute (26 h), short (3 days), medium (7 days) and long (14 days) fasting and periprandial changes on SgIIa mRNA level in hypothalamus, telencephalon and gut using Q-PCR method. The results showed that SgIIa mRNA increases under the effect of acute and short fasting, however, medium and long fasting did not affect SgIIa mRNA. Thirdly, we examined the effect of brain injection of goldfish SNa on food intake and locomotor behavior and the expression of some feeding neuropeptides such as neuropeptide Y, orexin, chocystokinin and cocaine-and amphetamine-regulated transcript I after treatment. Injection of SNa in the third brain ventricle increased food intake and fish activity. Associated with this was an increase in NPY and decrease in CARTI mRNA levels in hypothalamus. The increase in SgIIa mRNA following fasting and the increase of food intake as a result of SNa treatment suggest a novel role for SNa in feeding processes.

Des microparticules d’héparine de bas poids moléculaire (HBPM) ont été fabriquées suivant la méthode de la double émulsion à partir d’un polymère non biodégradable polycationique (Eudragit® RS) utilisé seul ou en mélange à différents pourcentages avec un polymère biodégradable (acide poly(lactique-co-glycolique)). Deux HBPM aux rapports anti-Xa/anti-IIa différents ont été testées : la nadroparine (3,6) et la tinzaparine (1,8). Les microparticules d’HBPM favorisent i) l’encapsulation des chaines longues actives (ACL) d’héparine par rapport aux chaines courtes actives (BCL) (diminution du rapport anti-Xa/anti-IIa) ii) la libération in vitro des chaines BCL (augmentation du rapport anti-Xa/anti-IIa), les chaines ACL étant fortement retenues au sein des microparticules. Cependant, la proportion de chaine BCL libérées par rapport à celle d’ACL est dépendante (cas de la nadroparine) ou non (cas de la tinzaparine) de la composition polymérique. Cette différence de comportement s’explique principalement par la capacité de l’Eudragit® RS à retenir les chaines ACL de nadroparine. Suite à l’administration orale de microparticules de nadroparine chez le lapin, une absorption des chaines ACL et BCL a été mise en évidence avec certaines formulations : dans ce cas, des rapports anti-Xa/anti-IIa plasmatique similaires à ceux résultant de l’injection sous-cutanée ont été obtenus, témoignant ainsi du potentiel de ces microparticules à remplacer la forme commerciale injectable. Lors d’études de localisation par microscopie confocale, le site d’absorption intestinal n’a pas été identifié. En revanche, des études de passage sur un épithélium issu de la culture cellulaire (Caco-2) semblent démontrer, contrairement aux résultats in vivo, que seules les chaines BCL de nadroparine seraient absorbées. Cependant, le mécanisme responsable du passage cellulaire reste à identifier. / Microparticles of low molecular weight heparin (LMWH) were prepared according to the double emulsion and extraction method using a non biodegradable polycationic polymer (Eudragit® RS) alone or blended according to different ratios with a biodegradable polymer (poly(lactic-co-glycolic) acid). Two LMWH presenting different anti-Xa/anti-IIa ratios were tested: nadroparin (3.6) and tinzaparin (1.8). LMWH microparticles facilitate i) the encapsulation of heparin active long chains (ACL) compared to active short chains (BCL) (anti-Xa/anti-IIa ratio decreased) ii) the in vitro release of short chains (anti-Xa/anti-IIa ratio increased) whereas long chains are held inside the microparticles. Nevertheless, when compared to ACL chains, the relative amount of BCL chains released is dependent (case of nadroparine) or not (case of tinzaparin) on the polymeric composition. This difference can be mainly due to the properties of Eudragit® RS to hold ACL chains of nadroparin. After an oral administration in rabbits of nadroparin microparticles, some formulations exhibited absorption of ACL and BCL chains: in this case, plasmatic anti-Xa/anti-IIa ratios in the same range than those observed following the subcutaneous injection were obtained. Such a result shows the potential of microparticles to be a good substitute of the commercial injectable dosage form, if the oral absorption is confirmed in human. During gastro-intestinal studies by confocal microscopy, the absorption site was not identified. Nevertheless, contrary to in vivo results, permeation studies on a cell cultured epithelium (Caco-2) demonstrated that BCL heparin chains were absorbed whereas ACL chains were not. However, the mechanism responsible of the cell permeation is still to be identified.

An understanding of plant pathogen-host interactions is essential to design efficient strategies to control disease in crops. Magnaporthe oryzae, an ascomyceteous fungus and causal agent of rice blast disease, is a model organism to study host-microbe interactions. The overall aim of this dissertation project was to identify genes involved in pathogenicity through the construction and characterization of a random insertional mutagenesis library. In order to saturate the genome with DNA inserts, a collection of >54,000 insertion lines of the M. oryzae strain 70-15 was generated via two transformation methods, PEG/CaCl2 (polyethylene glycol)-mediated protoplast transformation and Agrobacterium tumefaciens-mediated transformation. The first part of this dissertation describes the optimization of both transformation approaches, compares their efficiency and provides a description of the high-throughput processing and phenotypic analysis of the insertion lines. An in vitro appressorium assay of 12,000 T-DNA insertion strains allowed the identification of 135 lines that were classified as morphologically or functionally different than wild-type. Rice infection assays demonstrated that 112 of these strains exhibited defects in pathogenicity.The second part of this dissertation project analyzed the T-DNA integration patterns in a subset of pathogenicity mutants. This section aimed to identify the disrupted genes via recovery of M. oryzae sequences adjacent to the sites of T-DNA insertion. Genomic mapping of 61 T-DNA insertions in pathogenicity mutants via rescuing M. oryzae chromosomal T-DNA flanking sequences using inverse PCR resulted in the identification of 22 conserved hypothetical genes with predicted function, 11 predicted open reading frames without a GenBank significant match, two unannotated regions of the genome assembly and seven intergenic regions. The final part of this dissertation describes the characterization of a M. oryzae pathogenicity mutant that contains a T-DNA insertion in the upstream region of two divergently transcribed genes that encode the vacuolar type-ATPase subunit c`` and the general transcription factor TFIIA subunit γ. Genetic complementation demonstrated the insertion of the T-DNA in the promoter region of the general transcription factor TFIIA subunit γ is responsible for observed defects in conidiation, appressorium morphogenesis, and appressorium function. This is the first report relating the function of TFIIA subunit γ to pathogenicity.

Carnocyclin A (CclA) is a remarkably stable, potent bacteriocin produced by Carnobacterium maltaromaticum UAL307. Elucidation of the amino acid and genetic sequences revealed that CclA is a circular bacteriocin. Preliminary structural studies (dynamic light scattering, NMR, circular dichroism, stereochemical analysis) indicated that CclA is monomeric and alpha-helical in aqueous conditions and composed of L-residues. The 3D structure of [13C,15N]CclA was solved by NMR, revealing a compact arrangement of four helices. To examine the structure of the precursor peptide (pCclA) several fusion proteins were constructed and overexpressed; however, pCclA could not be isolated. To investigate the requirements for cyclization, several internally hexahistidine-tagged (His6) pCclA mutants were constructed. Expression conditions are underway. PisI was heterologously expressed and confirmed to impart protection against piscicolin 126 (PisA). Labeled and unlabeled PisA and PisI were purified following overexpression as maltose-binding protein fusions (MalE-fusions) and Factor Xa cleavage. NMR studies indicated that PisI and PisA do not physically interact. The 3D structure of PisI was solved by NMR, confirming that the four-helix bundle is a conserved motif for the immunity proteins of type IIa bacteriocins. The putative receptor proteins for these bacteriocins were cloned and overexpressed as His6-fusion proteins. Experiments are underway to optimize the expression and purification of these membrane proteins. The peptidase domain of the ABC-transporter protein (CbnTP) for carnobacteriocin B2 (CbnB2) was overexpressed as a His6-fusion protein. Active protease could not be purified from inclusion bodies, but was obtained as soluble protein following low-temperature overexpression. The CbnB2 precursor pCbnB2 (and a truncated derivative pCbnB2-RP) was purified following overexpression as a MalE-fusion and Factor Xa cleavage. pCbnB2 was incubated with CbnTP and MALDI-TOF and activity testing confirmed that CbnTP cleaved the leader peptide from pCbnB2. Five CysSer CbnTP mutants were constructed. Crystallographic studies of CbnTP are underway. Six bacteriocins (nisin, gallidermin, lacticin 3147, CclA, PisA, enterocin 710C) were tested against Gram-negative bacteria (E. coli DH5, Pseudomonas aeruginosa ATCC 14207, Salmonella typhimurium ATCC 23564) in the absence and presence of EDTA. PisA and lacticin 3147 exhibited minimal activity, whereas the other bacteriocins killed at least one strain, in the presence of EDTA.

Systemic lupus erythematosus (SLE) is a chronic multiorgan autoimmune disorder characterized by abundant immune complex (IC) deposition, with nephritis being a major cause of morbidity and mortality. Yet, IC deposition alone is not sufficient for disease development suggesting that additional factors dictate the propensity for developing target organ injury. Genome-wide association studies have identified polymorphisms in the leukocyte integrin Mac-1 (CD11b/CD18, ITGAM) that associate with lupus nephritis. Although Mac-1 promotes inflammation by triggering leukocyte recruitment and cytotoxic functions, there is emerging evidence that it may also serve protective roles under certain conditions. We demonstrate that Mac-1 deficiency in the context of the uniquely human FcgRIIA a receptor that binds IgG-IC, promotes susceptibility to lupus nephritis in two independent animal models. Analysis of renal tissue and intravital microscopy revealed that Mac-1 modulates neutrophil recruitment by FcgRIIA. The SLE-associated variant of Mac-1 rs1143679 (R77H), results in reduced Mac-1 functions, but the underlying mechanism remains undefined. CD18 integrin mediated adhesion is a multistep process that begins with affinity changes for ligand via transmission of allosteric signals. Moreover, mechanical forces (e.g. shear flow) paradoxically increase the lifetime of integrin-ligand bonds, referred to as "catch-bonds". Here, we show that expression of Mac-1 R77H on neutrophils, and blocking antibodies to the extracellular b-propeller domain in which it resides, markedly impairs Mac-1 adhesion to ligand under shear flow. R77H expressing cells exhibit a shift in equilibrium towards a bent conformation, a lower affinity and on- and off- rate for ligand and an inability to form catch-bonds. Additional mutants and activating antibodies reveal that R77H prevents allosteric signal transmission to the aI-domain required for productive ligand binding.

No description available.

This thesis addresses two separate problems - an investigation of the interaction of probe molecules with crystalline rutile and an investigation of the environment of group IA and IIA elements in organometallic compounds. Ab-initio Hartree-Fock calculations have been performed, aimed at investigating the interactions between the ionic surface of a crystal and an adsorbate molecule. Titanium dioxide, a material important for catalysis, electronic components and pigments, was chosen as the substrate, with carbon monoxide as the probe molecule. The calculations were carried out using the Crystal92 program, for the (110) surface of the Rutile polymorph of TiO(_2), employing a slab with a thickness of 5 atomic layers. The calculations investigated two orientations of the CO molecule with the molecular axis perpendicular to the surface. Results are reported showing contour diagrams for slices through the energy hypersurface parallel and perpendicular to the surface of the substrate. In order to facilitate the work described above, a program 'Builder2' was developed. This provides a convenient means for generating models of slabs of material from crystal structure data. Part of the development of Builder2 was to devise computer code to decompose standard Space Group symbols into the underlying symmetry matrices. The code for Builder2 is proprietary to Oxford Materials Ltd. and forms part of a commercial product. The environment of group IA and IIA elements in crystalline materials has not been the subject of any reported investigation. These elements, and organic ligands associated with them, play a significant role in biological systems. Around 16,000 atomic environments were extracted from the Cambridge Crystallographic Database to provide an up-to-date analysis of actual environments. The results are presented as histograms and tables, and suggestions are made for future extension of the analyses.

No description available.

Les mécanismes de formation des plaquettes sanguines à partir des mégacaryocytes ne sont pas totalement compris, mais l’environnement médullaire semble y avoir une influence cruciale. Dans ce travail nous montrons que i) les intégrines β3, récepteurs de protéines de matrice extracellulaire, semblent impliquées dans la mégacaryopoïèse et la formation des plaquettes, ii) la différenciation des cellules hématopoïétiques dans un environnement 3D de rigidité comparable à la moelle osseuse améliore la maturation des mégacaryocytes différenciés in vitro et iii) la myosine IIA est impliquée dans la distribution des organelles dans les mégacaryocytes. Parallèlement, Nous avons caractérisé la spécificité d’expression du transgène Pf4-cre pour valider son utilisation dans nos approches expérimentales. Ce travail apporte un éclairage nouveau sur le rôle de la myosine IIA et des intégrines dans les mégacaryocytes et souligne l’influence de la rigidité de l’environnement dans la mégacaryopoïèse. / Megakaryocytes differentiation (megakaryopoiesis) and platelet formation mechanisms are not entirely understood, but the bone marrow environment seems to be crucial in these processes. In this thesis, we show i) that integrin β3, the extracellular matrix protein receptors, are involved in megakaryopoiesis and platelet formation, ii) that recreating a 3D environment of stiffness in the range of that of bone marrow improves the maturation of in vitro differentiated megakaryocytes and iii) a new role for myosin IIA in the cytoplasmic distribution of organelles within the megakaryocyte. As a side-project, we characterized the specificity of expression of the Pf4-cre transgene to validate its use in our experimental approaches. This work enlightens new roles for myosin IIA and integrins in megakaryocytes and indicates that stiffness of the environment influences megakaryopoiesis.