Les cryptokarsts de Beez (Namur, Belgique) se sont développés au dépens de calcaires dolomitiques viséens, à la faveur de drains constitués par les filons sulfurés Fe-Pb-Zn, sous une couverture composée de pélites gréseuses viséo-namuriennes et de sables oligocènes. À Khamouda (Kasserine, Tunisie), les poches karstiques se sont développées suivant la stratification sub-horizontale des calcaires sénoniens de la Formation Douleb à partir d'une faille normale les mettant au contact de la couverture sableuse miocène (Formation Béglia). Dans les deux sites, des paragenèses riches et complexes s'installent aux interfaces entre encaissant carbonaté et remplissages karstiques. Des argilites blanches, principalement composées de phases silico-alumineuses et alumineuses, ainsi que des croûtes ferrugineuses sont toujours présentes. À Beez, l'argilite est constituée d'halloysite et de gibbsite. À Khamouda, elle s'enrichit de phases zincifères plus rares, telles que la sauconite, ainsi que d'un phyllosilicate à 7 Å et d'un hydroxyde de zinc amorphe. Des sulfates sont également néoformés, notamment du gypse, dissout par la suite mais dont les croûtes ferrugineuses comportent encore des indices, ainsi que de la jarosite à Beez. D'un point de vue fondamental, les systèmes cryptokarstiques, qui opposent une barrière carbonatée à la migration de fluides acides, sont des structures privilégiées pour l'étude et la compréhension de la migration et la fixation des éléments chimiques dans le domaine supergène. Dans les deux cas, les fluides météoriques acquièrent leur acidité (pH ~2 à Beez et pH~4 à Khamouda) par lessivage de la couverture sédimentaire sus-jacente et notamment par oxydation des sulfures qu'elle contient (pyrite à Beez, pyrite et sphalérite à Khamouda). Les principaux éléments mobilisés à Beez sont Si et Al et dans une moindre mesure Fe, Mn et les Terres Rares ; tandis qu'à Khamouda, les principaux éléments mobilisés sont Si, Al et Zn, et dans une moindre mesure Fe, Pb et les Terre Rares. La neutralisation des fluides au contact du mur carbonaté conduit dans un premier temps à la formation de sulfates (gypse, jarosite) et d'oxy-hydroxydes de fer, puis d'halloysite et d'hydroxydes d'aluminium à partir de pH 4,8-5,4. À Khamouda, les phases zincifères ne se forment que plus tardivement (pH < ~9,5). Dans les deux cas étudiés, nous avons mis en évidence l’évolution de phases minérales depuis des gels silico-alumineux jusqu’à des minéraux bien cristallisés, tels que l’halloysite, ou moins bien organisés, tels que certains oxydes de manganèse à Beez. Dans les deux gîtes, le microfaciès tubulaire de l'halloysite, correspondant à une croissance fissurale, prédomine par rapport au faciès sphéroïdal se développant habituellement au sein des masses de gel ; ce qui suggère une fracturation répétée des masses de gels précurseurs. Nous avons également établi que ces gels continuent à incorporer des cations des solutions percolantes, notamment du manganèse à Beez. La difficulté majeure de l'étude des altérations cryptokarstiques réside dans la détermination des âges des phénomènes. L'âge des couvertures sédimentaires impliquées dans les poches karstiques donne une première approximation. Ainsi, la phase majeure d'altération à Beez est post-oligocène suivie d'une réactivation quaternaire ; tandis que l'altération est post-miocène à Khamouda. Le site de Beez propose un éventail de minéralisations pouvant faire l'objet de datations radiométriques. Ces datations "absolues" doivent constituer une priorité forte à l'avenir. __________________________________________________________________________________________________ Résumé en anglais : The cryptokarsts from Beez (Namur, Belgium) were settled in dolomitic Visean limestones, in which vertical Fe-Pb-Zn sulphides veins play an important role as karstic drains. The sedimentary cover is made up of Viseo-Namurian siliceous shales and Oligocene sands. The cryptokarsts from Khamouda (Kasserine, Tunisia) were found in Senonian limestones (Douleb Formation). They expand from a down fault, which has brought limestones into contact with Miocene sands (Béglia Formation), following the sub-horizontal stratification. In both sites, complex paragenesis settled at the limestone/karst-filling interface. White clays, principaly composed of Si-Al and Al phases, and ferruginous crusts are the main paragenesis. In Beez, the white clays are made up of halloysite and gibbsite, while in Khamouda, they are enriched with uncommon zinciferous phases as sauconite (Zn-smectite), a 7Å-phyllosilicate and an amorphous Zn­hydroxide. Sulphates have also been found, as imprints of gypsum crystals in both sites, and as jarosite in Beez. Acid fluids percolated in the overlying sedimentary cover (pH~2 in Beez and pH~4 in Khamouda). The acidity is due to the oxidation of some sulphides (pyrite in Beez; pyrite and sphalerite in Khamouda). Cryptokarsts basically play an important role in chemical elements mobilization and trapping processes. In Beez, Si and Al have mainly been mobilized. Fe, Mn and the Rare Earth Elements (REE) have been mobilized too. In Khamouda, the main mobilized elements are Si, Al and Zn. Fe, Pb and REE have been mobilized too. The acid fluids are neutralized at the limestone karst-wall. It leads first to the neogenesis of sulphates (as gypsum and jarosite) and iron oxi-hydroxides (pH < 4,8). Then, halloysite and Al-oxi-hydroxides are formed (from pH~4,8-5,4 upward). In Khamouda, zinciferous phases developed lately (from pH~9,5 upward). In both studied systems, we clearly show the development of mineral phases from a Si-Al gel to well-crystallized minerals, as halloysite, or badly organized minerals, as some Mn-oxides from Beez. In both deposits, tubular halloysite, which usually develops in cracks, is prominent in comparison to spheroidal halloysite, which usually growths in gel masses. It suggests an extreme fracturing of the gel masses. We established that these gel masses mix cations from the percolating solutions. The main difficulty in cryptokarstic environments is to determine ages of weathering processes, nevertheless the age of overlying deposits give an idea. Thus, the major weathering stage in Beez is post-Oligocene (followed by a Quaternary reactivation), while weathering in Khamouda is post-Miocene. The cryptokarsts from Beez contain several mineral phases, which could be dated with radiometric methods. This "absolute" dating has to be the next step.

The cWnt activator, Rspondin-1 (Rspo1), has been identified as a regulator of β-cell growth and function, although its role in pathophysiological conditions such as streptozotocin (STZ)-induced diabetes is unknown. Hence, I hypothesized that Rspo1 deficiency stimulates β-cell neogenesis in STZ-diabetes. There was no difference in oral glucose handling between STZ-induced Rspo1mice, although, Rspo1-/- mice demonstrated increased insulin sensitivity compared to wild-type littermates. Moreover, β-cell mass and the total number of islets did not differ between STZ-induced Rspo1+/+ and Rspo1-/- mice, although mice with Rspo1 deficiency had reduced β-cell apoptosis and significantly enhanced numbers of insulin-positive ductal cells suggestive of β-cell neogenesis. Furthermore, the increased β-cell regeneration observed in knockout animals appeared to be associated with a more differentiated/mature β-cell phenotype in Rspo1-/- versus Rspo1+/+ mice. Collectively, these findings indicate a role for Rspo1 as a negative regulator of in vivo β-cell neogenesis and survival in the face of STZ-induced diabetes.

The cWnt activator, Rspondin-1 (Rspo1), has been identified as a regulator of β-cell growth and function, although its role in pathophysiological conditions such as streptozotocin (STZ)-induced diabetes is unknown. Hence, I hypothesized that Rspo1 deficiency stimulates β-cell neogenesis in STZ-diabetes. There was no difference in oral glucose handling between STZ-induced Rspo1mice, although, Rspo1-/- mice demonstrated increased insulin sensitivity compared to wild-type littermates. Moreover, β-cell mass and the total number of islets did not differ between STZ-induced Rspo1+/+ and Rspo1-/- mice, although mice with Rspo1 deficiency had reduced β-cell apoptosis and significantly enhanced numbers of insulin-positive ductal cells suggestive of β-cell neogenesis. Furthermore, the increased β-cell regeneration observed in knockout animals appeared to be associated with a more differentiated/mature β-cell phenotype in Rspo1-/- versus Rspo1+/+ mice. Collectively, these findings indicate a role for Rspo1 as a negative regulator of in vivo β-cell neogenesis and survival in the face of STZ-induced diabetes.

abstract: Though for most of the twentieth century, dogma held that the adult brain was post-mitotic, it is now known that adult neurogenesis is widespread among vertebrates, from fish, amphibians, reptiles and birds to mammals including humans. Seasonal changes in adult neurogenesis are well characterized in the song control system of song birds, and have been found in seasonally breeding mammals as well. In contrast to more derived vertebrates, such as mammals, where adult neurogenesis is restricted primarily to the olfactory bulb and the dentate gyrus of the hippocampus, neurogenesis is widespread along the ventricles of adult amphibians. I hypothesized that seasonal changes in adult amphibian brain cell proliferation and survival are a potential regulator of reproductive neuroendocrine function. Adult, male American bullfrogs (Rana catesbeiana; aka Lithobates catesbeianus), were maintained in captivity for up to a year under season-appropriate photoperiod. Analysis of hormone levels indicated seasonal changes in plasma testosterone concentration consistent with field studies. Using the thymidine analogue 5-bromo-2-deoxyuridine (BrdU) as a marker for newly generated cells, two differentially regulated aspects of brain cell neogenesis were tracked; that is, proliferation and survival. Seasonal differences were found in BrdU labeling in several brain areas, including the olfactory bulb, medial pallium, nucleus accumbens and the infundibular hypothalamus. Clear seasonal differences were also found in the pars distalis region of the pituitary gland, an important component of neuroendocrine pathways. BrdU labeling was also examined in relation to two neuropeptides important for amphibian reproduction: arginine vasotocin and gonadotropin releasing hormone. No cells co-localized with BrdU and either neuropeptide, but new born cells were found in close proximity to neuropeptide-containing neurons. These data suggest that seasonal differences in brain and pituitary gland cell neogenesis are a potential neuroendocrine regulatory mechanism. / Dissertation/Thesis / M.S. Biology 2012

The accumulation of leukocytes in non-lymphoid tissues and their structural organization into tertiary lymphoid structures (TLS), a process known as ectopic lymphoid neogenesis (ELN), is observed in response to chronic inflammation and in the target organ of several autoimmune diseases. TLS strongly resemble secondary lymphoid organs with specialised high-endothelial venules (HEV), segregated B/T cell areas and presence of follicular dendritic cells (FDC) networks promoting in situ affinity maturation of the antibody response. TLS have been associated with a growing number of autoimmune conditions and usually their presence is prognostic for undesirable disease progression. In Sjögren’s syndrome (SS), an autoimmune disease affecting the salivary and lachrymal glands leading to exocrine dysfunction, TLS develop in the salivary glands (SG) of around one-third of the patients. The immunobiology of the SG and the pathogenesis of SS have been poorly clarified and to date a robust and reproducible inducible animal model of SS and TLS in the SG is still absent. In my PhD, I developed and validated a novel inducible model of ELN in murine SG that also reproduces several features of SS. The retrograde administration of a replication-deficient adenovirus (AdV) in the SGs of wild-type C57Bl/6 mice was able to induce within three weeks fully formed TLS that displayed B/T cell segregation, FDC networks, HEVs and were positive for markers of germinal centres. Moreover, the AdV-treated mice showed a significant reduction of salivary flow and in 75% of the cases development of anti-nuclear antibodies.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23100号 / 医博第4727号 / 新制||医||1050(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 平井 豊博, 教授 河本 宏, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Hair follicle (HF) regeneration can be achieved in the center of large full-thickness wounds on mouse backs (wound-induced HF neogenesis model, WIHN). Investigations with this model have allowed for the identification of some of the factors limiting the extent of fibrosis, which creates a permissive environment for the reposition of HF. For WIHN, specific subpopulations of cells rather than cell types are permissive to this process. Detailed information on the cellular composition in WIHN is not available. Here, we provide a description of changes in cell numbers of fibroblasts, HF dermal papilla, endothelial cells, keratinocytes (interfollicular epidermis, HF-infundibulum, HF-isthmus, HF-bulge (basal and suprabasal), HF-hair germ) and immune cells (macrophages, monocytes, dendritic cells, T cells (CD4+ , CD8+ , CD4+/CD8+ , regulatory T cells) and neutrophils) based on flow cytometric analysis. We compared unwounded skin with large wounds (1.5 × 1.5 cm) at different time points after wounding. We found that non-immune dermal cells have the largest share in the skin at all time points studied, and that the number of epidermal cells started increasing nine days after wounding, which precede isthmus cells and bulge cells, mirroring the development of hair follicles. Monocytes and neutrophils represent most myeloid cells in wounds and remain in wounds even beyond the inflammatory phase of wound healing. Macrophages can be identified as inflammatory and alternative cells and are also found in wounds even in the late remodeling phase of wound healing. Lastly, we provide information about T cells in large wounds. Most T cells in the wounds were CD8+ at all time points and expressed γδTCR, which was previously thought to be expressed mainly on CD4+ . We also report the existence of double positive CD4/CD8. Our study provides a guide in terms of time points suitable for the further study of cell subpopulations aiming to dissect the cellular heterogeneity in WIHN. Our results might set the base for the comparison of WIHN between control mice and animals manipulated to influence HF neogenesis and the full understanding of the responsible actors allowing for HF regeneration.

Type 1 diabetes is the direct result of an autoimmune attack on the pancreatic islet cells. The islets contain b cells, which are the only type of cell capable of supplying insulin in the human body. The destruction of these cells leaves the diabetic to rely on exogenous insulin to maintain a normal blood sugar level. Insulin therapy allows the diabetic to deal with the symptoms of the disease, but does nothing for the underlying condition. In order to truly cure the disease, the strategy is to replenish the b cells in the diabetic. Islet neogenesis associated protein (INGAP) has been shown to regenerate islet cells and reverse experimentally-induced diabetes in animal models. The INGAP pentadecapeptide is a 15 amino acid peptide from INGAP with comparable activity to the full-length protein. This 15-mer is undergoing clinical trials for treating diabetes. The overall goal of the project described in this work is to determine the structure of the INGAP pentadecapeptide for use in structure-based drug design of non-peptide mimics of the 15-mer. The first set of experiments in the present work directly examined the 15-mer in solution using NMR. No stable structure of the small peptide was found. The second set of experiments involved a homolog of INGAP, called INGAP-related protein, or INGAPrP. INGAPrP was recombinantly produced in E. coli and subsequently purified and refolded. Refolding of INGAPrP was verified by a 1H-15N HSQC experiment. CD experiments supported the NMR study, indicating helical content in INGAPrP. The folded nature of the protein will allow for the three-dimensional structure of INGAPrP to be determined. The protein structure will show the fold of the 15-mer within the full-length protein. This information will be valuable for the ultimate goal of producing structural mimics of the INGAP pentadecapeptide. Non-peptide mimics should have better oral bioavailability and longer half-lives in vivo.

Introduction: les follicules lymphoïdes (FL) sont absents du poumon normal mais ont été décrits dans les poumons de patients atteints de mucoviscidose ou de dilatations de bronches non mucoviscidosiques, suggérant un rôle pour l’infection bronchique dans la néogenèse lymphoïde (NL). Nous avons étudié la dynamique de la néogenèse lymphoïde dans l’infection bactérienne. Méthodes: les souris C57BL/6 ont reçu une instillation intratrachéale de billes d’agarose contenant du PAO1 ou du S. aureus (106 CFU/animal) permettant une infection prolongée et ont été comparées à des souris contrôles (billes stériles ou absence de billes). Les souris ont été sacrifiées à J1, J4, J7 et J14. Résultats: l’instillation unique de billes d’agarose contenant du PAO1 ou du S. aureus induit en 14 jours des FL fonctionnels situés sous l’épithélium en regard des zones d’infection. Le marquage pour CXCL12 et CXCL13 est faible chez contrôles, mais présent dans l’épithélium (CXCL13) dès J1 et présent également dans les FL (CXCL12 et CXCL13) à J14 chez les souris infectées. Le traitement des souris par un anticorps anti CXCL12 ou anti CXCL13 n’inhibe pas la formation des FL induite par l’infection à PAO1. Conclusion: nos données suggèrent un rôle pour l’infection bactérienne prolongée et l’épithélium respiratoire dans la NL des bronchopathies chroniques. Notre modèle permet d’évaluer les mécanismes de la formation et de persistance des FL dans le poumon. / Introduction: lymphoid follicles (LF) are absent in normal lungs, but are described in lungs of subjects with cystic fibrosis (CF) or non-CF bronchiectasis, suggesting a role for bacterial infection in lymphoid neogenesis. We aimed to study the dynamic of pulmonary lymphoid neogenesis (LN) during bacterial infection. Methods: C57BL/6 mice were instilled intratracheally with PAO1- or S. aureus-coated (1.106 CFU/mouse) agarose beads (which produced prolonged airway infection) and compared to controls (sterile beads or no instillation). Mice were sacrificed on day (d)1, d4, d7, and d14 after instillation. Results: chronic pulmonary infection with PAO1 or S. aureus induced organised LF in 14 days after a single challenge with PAO1- or S. aureus-coated beads. Bacteria- induced LF were exclusively localized in the subepithelium of infected airways. Staining for CXCL12 and CXCL13 was weak in airway epithelium of controls, but was positive in airway epithelium (CXCL13) at 1 day and in LF (both) of infected mice at 14 days. Treatment with anti CXCL12 or anti CXCL13 Ab did not reduce LN induced by PAO1 infection. Conclusions: chronic bacterial infection and respiratory epithelium could contribute to LN in chronic airway diseases. Our unique model allows to study mechanisms for the formation and maintenance of lung LF.