Les acteurs neuroendocriniens régulant la physiologie des Lophotrochozoaires, groupe frère des Ecdysozoaires parmi les Protostomiens, demeurent peu connus. Grâce à l’émergence récente de ressources génomiques, transcriptomiques et peptidomiques chez l’huître creuse Crassostrea gigas, l’étude des couples ligand(s)/récepteur(s) régulant les fonctions physiologiques est désormais facilitée. Ainsi, par une approche d’endocrinologie inverse consistant à éprouver l’activité d’un panel de ligands potentiels, plusieurs récepteurs couplés aux protéines G (RCPGs), jusqu’alors orphelins ont pu être caractérisés sur le plan fonctionnel. Trois voies de signalisation ont été étudiées : la voie de type cholécystokinine/sulfakinine (CCK/SK) la voie de type calcitonine (CT) et la voie de type dopamine (DA). Grâce à des tests fonctionnels, deux neuropeptides et deux récepteurs de type CCK/SK ont pu être caractérisés. Des tests d’activité biologique in vitro et des expériences de conditionnement alimentaire ont montré la potentielle implication de ces couples dans la régulation de la digestion et de la satiété chez l’huître. Par ailleurs, deux couples neuropeptide/récepteur de type CT ont été caractérisés montrant, à l’image de leurs homologues chez les vertébrés, leur possible rôle dans la régulation hydrique ou ionique. D’autre part, un récepteur activé de manière spécifique par la dopamine et la tyramine a été caractérisé. Ce système de signalisation semble être impliqué dans la médiation du stress et intervenir dans les processus régulateurs de la reproduction au niveau de la gonade. Ainsi, les différents résultats obtenus au cours de ces travaux ont permis d’identifier des couples ligands/récepteurs d’huître homologues de systèmes de signalisation présents chez les Ecdysozoaires et les vertébrés confirmant l’origine de ces systèmes neuroendocriniens depuis l’ancêtre commun des Bilatériens. Les résultats obtenus ont également permis de mieux comprendre comment l’huître intègre les paramètres du milieu et donc s’acclimate aux différentes contraintes environnementales. / The neuroendocrine regulators of the physiology of Lophotrochozoa, the sister clade of Ecdysozoa among Protostoma, remain poorly understood. Thanks to the recent emergence of genomic, transcriptomic and peptidomic resources in the Pacific oyster Crassostrea gigas, the functional characterization of ligand/receptor pairs regulating a diversity of physiological functions has been facilitated. Using a reverse endocrinology approach, a number of orphan G Protein-Coupled Receptors (GPCRs) have been functionally characterized. Three signalling systems have been studied in the oyster: The cholecystokinin/sulfakinin (CCK/SK), the calcitonin (CT) and the dopamine (DA) signalling systems. Two CCK/SK receptors and ligands have been characterized. In vitro bioassays and feeding conditions suggested the potential involvement of this signalling system in the regulation of digestion and satiety. Besides, two couples of CT-type peptides and receptors have been characterized showing, as for their vertebrate counterparts, their possible role in the regulation of water and ion balance. A receptor specifically activated by dopamine and by tyramine has also been characterized. This signalling system appeared to be implicated in the mediation of stress and to play a role in the regulatory processes of reproduction in the gonads. This study allowed the characterization in the oyster of ligand receptor pairs homolog to known signalling systems present in Ecdysozoa and vertebrates, thus confirming the origin of these neuroendocrine systems in the common ancestor of Bilateria. The results of this study also contributed to understand how the oyster integrates external parameters and adapts to various environmental constrains.

The activation of 1,25D-dihydroxyvitamin D3 (1,25D) is catalysed by the enzyme 25-hydroxyvitamin D-1ƒhydroxylase (CYP27B1) in the kidney, which is the primary producer of 1,25D in the body. Although the synthesis of 1,25D by CYP27B1 and the catabolism of 1,25D by 25-hydroxyvitamin D-24-hydroxylase (CYP24) also take place in the bone, the significance of the bone cell-specific metabolism of vitamin D remains largely unknown. This thesis investigates the regulation of the expression of CYP27B1, CYP24 and vitamin D receptor (VDR) mRNA, both in the bone and in the kidney, with the aim to determine whether the regulation of the vitamin D metabolism in the bone is independent from that in the kidney. The effects of age, dietary calcium and vitamin D status on the expression these genes in both the kidney and the bone, as well as on a number of biochemical factors known to regulate the renal metabolism of 1,25D, such as PTH, calcium and 1,25D itself, were examined. CYP27B1 mRNA expression was also studied in histological sections of rat femoral bone. Furthermore, CYP27B1, CYP24 and VDR mRNA expression were also identified in specific regions of the rat femur and in a number of bone cell lines, with the aim to identify the bone cell types that have the capacity to metabolise and/or to respond to vitamin D. The age-related decrease in the circulating levels of 1,25D detected in animals ranging in age from 3 weeks to 2 years old, was a direct result of a reduction in the expression of CYP27B1 mRNA and an increase in the expression of CYP24 and VDR mRNA in the kidney. In contrast, the expression of CYP27B1 and CYP24 mRNA in the bone is high from 3 to 15 weeks of age, which is the period of rapid growth and development. The expression of CYP27B1 mRNA in the bone was positively correlated with the circulating levels of calcium throughout aging, which suggests that the 1,25D produced in the bone may be involved in the mineralisation process. The positive correlation found between the expression of CYP27B1 and CYP24 mRNA in the bone was in contrast with the negative correlation found between the expression of these two enzymes in the kidney. This suggests that the 1,25D produced locally in the bone, rather than the 1,25D produced in the kidney, is the primary determinant of the CYP24 activity in the bone. In vitamin D-deplete animals, fed a 0.1% calcium diet (D(-)/LC), the expression of CYP27B1 mRNA was induced and the expression of CYP24 mRNA was suppressed in the kidney. In contrast, both the expression of CYP27B1 and CYP24 mRNA were low in the bones of these D(-)/LC animals. When vitamin D-deplete animals were fed a 1% calcium diet (D(-)/HC), the expression of both CYP27B1 and CYP24 mRNA was high in the bone, which was in direct contrast with the low expression of these genes detected in the kidney. Besides this, a positive correlation was found between the expression of CYP27B1 mRNA in the bone, serum calcium levels and bone mineral volume (BV/TV) in the epiphysis, which supports the findings for the age study that the locally produced 1,25D may be involved in the promotion of bone mineralisation. Although serum PTH levels was positively correlated with the expression of CYP27B1 mRNA in the kidneys of hypocalcaemic animals, there was no such relationship detected between the levels of serum PTH and the expression of CYP27B1 mRNA in the bone. This finding suggests that the regulation of the expression of CYP27B1 mRNA in the bone is different from the regulation found in the kidney. The identification of CYP27B1 mRNA in osteoblasts-like cells, taken together with the associations between serum calcium and CYP27B1 mRNA expression in the previous studies, suggests that 1,25D produced in osteoblasts may play a significant role in the bone mineralisation process. The detection of CYP27B1 mRNA expression in a number of bone marrow cells suggests that locally produced 1,25D may also play a role in the growth and differentiation of hematopoietic cells. / Thesis (Ph.D.)--School of Molecular and Biomedical Science, 2003.

Peptidases regulate important physiological processes by controlling levels of bioactive peptides and occasionally through noncatalytic processes. This thesis presents a study of prolyl endopeptidase-like (PREPL), which is a peptidase involved in several human deletion syndromes, including hypotonia-cystinuria syndrome (HCS). Phenotypes tentatively attributed to PREPL deletion include hypotonia and decreased growth hormone (GH) levels. However, little is known about the mechanisms by which PREPL deletion causes these phenotypes. To better understand PREPL catalytic activity, we used an activity-based protein profiling fluorescence polarization screen to identify the first specific PREPL inhibitors. We proceeded to demonstrate the activity of these inhibitors in cells and discovered several classes of cell-active PREPL inhibitors. Further, one of these inhibitors, 1-isobutyl-3-oxo-3,5,6,7-tetrahydro-2H-cyclopenta[c]pyridine-4-carbonitrile, was able to enter mouse brains. To characterize PREPL substrate specificity, we performed several substrate profiling experiments, but no substrates could be identified, in line with reports from other groups who used related approaches to attempt to identify PREPL substrates. To characterize any noncatalytic functions of PREPL, we used an affinity purification-mass spectrometry approach (AP-MS) to search for any protein-protein interactions of PREPL. We identified brain-expressed X-linked 2 (BEX2) as a novel interactor of PREPL, and confirmed this interaction by immunoblot. Several other proteins identified in the AP-MS experiment, including several members of the STRIPAK complex are being further investigated for possible PREPL interaction. To determine whether HCS phenotypes are in fact due to PREPL deletion and to delineate the molecular pathways involved, we generated a conditional PREPL knockout mouse. These mice were visibly smaller than wildtypes and growth curve analysis verified that from week three of life, there was a significant difference in weight between wildtype and knockout mice. Initial surface righting task experiments also indicate that PREPL knockout pups may have a hypotonia phenotype. In summary, we have developed several new tools for studying PREPL catalytic and noncatalytic function, demonstrated that PREPL deletion causes a GH-related growth deficiency and possible hypotonia and thus moved several steps closer to understanding the molecular mechanisms underlying PREPL deletion phenotypes. / Chemistry and Chemical Biology

Advances in biotechnology and recombinant technologies have lead to the production of several classes of new drugs such as peptide and protein drugs. These compounds are mostly indicated for chronic use but their inherent characteristics such as size, polarity and stability prevent them from incorporation in novel dosage forms. The bioavailability of nearly all peptide drugs is very low due to poor absorption from the administration site. Several challenges confront the pharmaceutical scientist in developing effective and innovative dosage forms for these classes of drugs. A lot of attention has been given to the nasal route of drug administration for delivery of peptide drugs. The availability of several promising classes of absorption enhancers and new drug delivery technologies has also prompt scientists to develop new delivery systems for nasal administration of peptide drugs. It has been shown in recent years that N-trimethyl chitosan chloride (TMC), a quaternary derivative of chitosan, is effective in enhancing the absorption of several peptide drugs, both in the peroral route and in the nasal route of drug administration. Early indications are that new drug delivery technologies such as Pheroid technology will also be able to enhance peptide drug absorption in the nasal route. The aim of this study was to evaluate and compare the absorption enhancing abilities of TMC and Pheroid technology in the nasal delivery of calcitonin, a peptide hormone with low bioavailability. Pheroid vesicles and Pheroid microsponges were prepared and characterized for their morphology and size distribution. Calcitonin was entrapped into these vesicles and microsponges and TMC and TMO solutions (0.5 % w/v), containing calcitonin, was also prepared. These formulations were administered nasally to rats in a volume of 100 μl/kg body-weight to obtain a final concentration of 10 IU/kg body-weight of calcitonin. Plasma calcitonin and calcium levels were determined over a period of 3 hours. The results of this study clearly indicated that both Pheroid formulations and the TMC formulation increase the nasal absorption of calcitonin with a resulting decrease in plasma calcium levels, indicating an increased absorption of calcitonin. The highest increase in calcitonin absorption was obtained with the TMC formulation and this was explained by the difference in the mechanism of action in enhancing peptide absorption between TMC and Pheroid technology. It was concluded that Pheroid technology is also a potent system to enhance peptide drug delivery and that the exact mechanism of action should be investigated further. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2006.

Change particularities of the blood serum levels of vitamin D, calcitonin and parathyroid hormone were analysed in healthy cows of different feeding, age and productivity and in sick cows with metabolic diseases. It was investigated how biochemical indicators vary in cows with metabolic diseases (parturient paresis, osteomalacia and mastitis) and the most informative indicators for diagnostics of these diseases (for levels of calcium, phosphorus and magnesium) were determined. Obtained findings were processed statistically. The method of electrochemiluminescence analysis used for analysis of the blood serum levels of PTH in humans was applied. Compared with radioimmunic method, this method is not expensive and quite fast; in addition, this method is sensitive and reliable, because it permits to detect low levels of PTH in the blood serum (up to 0.127 pmol/l). The method of chemiluminescence immunometric analysis used to find the level of CT in humans was tested. Also the method of immunoferment analysis (ELISA) was tested to measure the blood serum level of 25-OH vitamin D in cattle using human antibodies.

Advances in biotechnology and recombinant technologies have lead to the production of several classes of new drugs such as peptide and protein drugs. These compounds are mostly indicated for chronic use but their inherent characteristics such as size, polarity and stability prevent them from incorporation in novel dosage forms. The bioavailability of nearly all peptide drugs is very low due to poor absorption from the administration site. Several challenges confront the pharmaceutical scientist in developing effective and innovative dosage forms for these classes of drugs. A lot of attention has been given to the nasal route of drug administration for delivery of peptide drugs. The availability of several promising classes of absorption enhancers and new drug delivery technologies has also prompt scientists to develop new delivery systems for nasal administration of peptide drugs. It has been shown in recent years that N-trimethyl chitosan chloride (TMC), a quaternary derivative of chitosan, is effective in enhancing the absorption of several peptide drugs, both in the peroral route and in the nasal route of drug administration. Early indications are that new drug delivery technologies such as Pheroid technology will also be able to enhance peptide drug absorption in the nasal route. The aim of this study was to evaluate and compare the absorption enhancing abilities of TMC and Pheroid technology in the nasal delivery of calcitonin, a peptide hormone with low bioavailability. Pheroid vesicles and Pheroid microsponges were prepared and characterized for their morphology and size distribution. Calcitonin was entrapped into these vesicles and microsponges and TMC and TMO solutions (0.5 % w/v), containing calcitonin, was also prepared. These formulations were administered nasally to rats in a volume of 100 μl/kg body-weight to obtain a final concentration of 10 IU/kg body-weight of calcitonin. Plasma calcitonin and calcium levels were determined over a period of 3 hours. The results of this study clearly indicated that both Pheroid formulations and the TMC formulation increase the nasal absorption of calcitonin with a resulting decrease in plasma calcium levels, indicating an increased absorption of calcitonin. The highest increase in calcitonin absorption was obtained with the TMC formulation and this was explained by the difference in the mechanism of action in enhancing peptide absorption between TMC and Pheroid technology. It was concluded that Pheroid technology is also a potent system to enhance peptide drug delivery and that the exact mechanism of action should be investigated further. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2006.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

Diss. (sammanfattning) Linköping : Univ., 2001. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 12 uppsatser.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2006. / Härtill 4 uppsatser.