The current study investigated the distribution of periodontal epithelial cells and nerve fibres within the furcations of rat maxillary molar teeth subjected to hypothermic injury. The upper right first molars of 30 Sprague-Dawley rats were subjected to a single 20 minute application of dry ice in order to produce aseptic necrosis within the periodontal ligament, while the contralateral first molar served as an untreated control. Five animals were each sacrificed via cardiac perfusion after 7, 10, 14, 18, 21 and 28 days respectively and the maxillae were dissected out. After fixation in paraformaldehyde and processing, the tissues were embedded in paraffin wax and cut into 7µm serial coronal sections through the furcation region. Consecutive sections were then stained with H&E, cytokeratin AE1/AE3 and PGP 9.5 immunostains. Light microscopic examination of the H&E stained sections revealed that ankylosis had not developed in all of the experimental teeth, and in some of the observation groups fewer teeth were ankylosed than unaffected. The morphology of the ankylotic areas appeared to change with time, initially consisting of fine bony trabeculae, then progressing to solid bone occupying the entire furcation before becoming less solid again by the latest observation periods. Root resorption was often seen adjacent to areas of ankylosis, but the cementum of the tooth root at the point of ankylotic union was usually intact and free of resorption. Changes within the pulp chambers of the experimental teeth were also noted, with reduction in cellularity and tissue disorganisation initially, then increasing cellularity and formation of a cementum-like material on the chamber walls later. Cytokeratin AE1/AE3 immunostaining successfully identified epithelial cells within the periodontal ligament and their distribution around control teeth was similar to previous reports. Counting of these cells revealed lower numbers around experimental teeth, with the lowest counts around experimental teeth which had developed ankylosis. No change in the epithelial cell counts was detected over time, and these cells did not appear to regenerate after necrosis regardless of whether or not ankylosis developed. Statistical analysis indicated that the probability of ankylosis decreased as the number of epithelial cells increased. The PGP 9.5 immunostain identified periodontal nerve fibres, but the use of this stain was quite technique sensitive. The furcations of the molar teeth were noted to have relatively sparse innervation, with most of the visible nerve fibres being closely associated with blood vessels and located in the outer two-thirds of the ligament. Counting of the nerve fibres revealed fewer fibres around experimental teeth compared to control teeth, especially in the part of the ligament closest to the tooth root. There was no relationship detected between nerve count and time or between nerve and epithelial cell counts. Resorption was found to be more prevalent in experimental teeth, and the probability of resorption in a given tooth decreased as the epithelial cell count increased. The findings of this study suggest that the epithelial cells within the periodontal ligament have a protective function in the prevention of dentoalveolar ankylosis and resorption. Evidence of an intimate interrelationship between periodontal nerve fibre and epithelial cell numbers could not be confirmed. The null hypothesis that epithelial cell rests of Malassez do not provide a protective function against ankylosis and external root resorption was rejected, and the null hypothesis that nerve fibres and epithelial cells are not inter-dependent was retained. / http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1297409 / Thesis (D.Clin.Dent.) -- School of Dentistry, 2007

[Truncated abstract] This study describes the results of a four and a half year investigation examining local regulation of ion transport through pumping epithelial cells. The study focussed on the standard isolated toad skin preparation, made famous by Hans Ussing. Originally, the objective was to perform some simple manipulations on the isolated toad skin, a standard and well-tested epithelial layer, which, according to the literature, was a well-behaved and stable preparation. The purpose of doing these toad skin experiments was to gain familiarity with the experimental techniques, such as measuring the open-circuit voltage (Voc) and the short-circuit current (Isc) across an epithelium. In the process, the experimental information that was obtained was to assist in the development and refinement of a mathematical model of a single pumping epithelial cell . . . Finally, it should be emphasised the toad skin was a convenient tissue model for exploring more general issues such as: (i) how pumping epithelial cells may adjust to changes in the extracellular environment by locally regulating their membrane conductances; (2) how the topology of a cell can influence its function (i.e. the topology can determine whether a cell is optimised for salt transport or water transport). (3) how different cells, with different functions, may be positioned in apposition in a pumping epithelial tissue so that gradients generated by one cell type can be utilised by another. From a broader perspective, it is likely that such issues are also applicable to other pumping epithelia, and ultimately, may assist in understanding how these epithelia function.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 191-209).

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2007. / Härtill 4 uppsatser.

Thesis (Ph.D. in Toxicology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 130-174).

Orientador: Janete Dias Almeida / Coorientadora: Celina Faig Lima Carta / Banca: César Augusto Cardoso / Banca: Antonio José Gonçalves / Banca: Yasmin Rodarte Carvalho / Banca: Luiz Eduardo Blumer Rosa / Resumo: Tabaco e álcool são considerados os principais fatores de risco para o carcinoma de células escamosas (CCE) bucal contribuindo de maneira desfavorável para o tratamento e desfecho clínico. Seus carcinógenos são metabolizados em duas fases, sendo a segunda fase realizada pelas Glutationa S-transferases (GSTs). O objetivo do presente estudo foi avaliar a expressão gênica da forma selvagem dos genes GSTM1, GSTP1 e GSTT1 por qPCR em 33 amostras de CCE bucal de fumantes, ex-fumantes e não fumantes, e 15 controles em busca de uma correlação clínica com consumo de tabaco, álcool e estadiamento clínico. A dependência nicotínica foi avaliada pelo Teste de Fagerström pra Dependência a Cigarros (TFDC) e para consumo de etílicos o Teste AUDIT. Foi observado aumento da expressão de GSTM1 no Grupo CCE fumante em relação ao Grupo Controle (p=0,0161). Contrariamente, foi encontrada uma menor expressão de GSTT1 no Grupo CCE fumante em relação ao Grupo Controle fumante (p=0,0183). No grupo CCE fumante não foi encontrada uma correlação entre a expressão dos genes estudados e fatores ligados ao tabagismo, etilismo e estadiamento clinico. No grupo Controle fumante, houve correlação entre teste AUDIT e a expressão de GSTM1 (p=0,0000). Para GSTP1 e GSTT1 houve correlação entre a expressão quando comparada a idade do paciente (p=0,0008; p=0,0095), idade de inicio do tabagismo (p=0,0033; p=0,0081), TFDC (p=0,0102; p=0,0085) e AUDIT (p=0,0052; p=0,0219) respectivamente. Para GSTT1 foi encontrada uma c... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Tobacco and alcohol are considered to be the main risk factors for oral squamous cell carcinoma (SCC), contributing to treatment and clinical outcome. Its carcinogens are metabolized in two phases, being the second phase carried out by Glutathione Stransferases (GSTs). The objective of the present study was to evaluate the wild-type gene expression of the GSTM1, GSTP1 and GSTT1 genes by qPCR in 33 samples of oral SCC from smokers, former smokers and nonsmokers, and 15 controls looking for a clinical correlation with tobacco and alcohol consumption and clinical staging. Nicotinic dependence was assessed by the Fagerström Test for Cigarette Dependence (TFCD) and alcohol consumption by the AUDIT Test. Increased expression of GSTM1 in the Smoker SCC Group was observed in relation to the Control Group (p=0.0161). Conversely, a lower expression of GSTT1 was found in the smoker SCC group compared to the Smoker Control Group (p=0.0183). In the smoker SCC group, no correlation was found between the genes expression studied and factors related to smoking, alcoholism and clinical staging. In the Smoker Control Group, there was a correlation between the AUDIT test and the GSTM1 expression (p=0.0000). For GSTP1 and GSTT1, there was a correlation between the expression compared with the patient's age (p=0.0008, p=0.0095), age of starting smoking (p=0.0033, p=0.0081), FTCD (p=0.0102, p=0.0085) and AUDIT (p=0.0052, p=0.0219) respectively. For GSTT1 a correlation was found between expression ... (Complete abstract click electronic access below) / Doutor

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Les N-acyl homosérine lactones sont des molécules du quorum sensing impliquées dans la communication interbactérienne mais elles sont également capables d'intéragir avec les cellules eucaryotes. Rechercher ces molécules dans le contexte des maladies inflammatoires chroniques intestinlaes (MICI) et plus particulièrement dans le cadre de l'étude des conséquences de la dysbiose sur les voies de l'inflammation intestinale est séduisant. En utilisant la spectrométrie de mase, nous avons mis en évidence pour la première fois des AHLs dans l'écosystème intestinal humain, et plus particulièrement une nouvelle AHL, 3-oxo-C12 :2, qui est prédominante. Cette AHL est corrélée à la normobiose, est perdue au cours des MICI et exerce un effet protecteur sur les cellules épithéliales intestinales. En effet, la 3-oxo-C12 :2 exerce un effet anti-inflammatoire in vitro sur les cellules Caco-2 sans augmenter la perméabilité paracellulaire. De plus, les premiers résultats in vivo montrent que la 3-oxo-C12 est également capable d'influencer la composition du microbiote intestinal des souris. Ces résultats ouvrent de nombreuses perspectives notamment dans la recherche de traitements écologiques au cours des MICI. / Quorum sensing molecules N-acyl-homoserine lactones (AHLs) involved in bacterial communication network are also able to interact with eukaryotic cells. Searching for these molecules in the context of inflammatory bowel diseases (IBD) and more precisely when studying consequences of dysbiosis on gut inflammation pathways is appealing. Using mass spectrometry, we identified for the first time AHLs in human intestinal ecosystem, and among them a new AHL, 3-oxo-C12:2 which is prominent. This AHL correlates with normobiosis, is lost IBD and exerts protective effect on gut epithelial cells. In fact, 3-oxo-C12:2 exerts anti-inflammatory effect in vitro on Caco-2 cells without increased paracellular permeability. Furthermore, first results from in vivo experiments show that 3-oxo-C12:2 is also able to influence mice gut microbiota composition. These results open multiple perspectives especially on new ecological treatments in IBD.

Les cellules souches hématopoïétiques (CSH) assurent la génération de toutes les lignées sanguines. Leur différenciation en cellules T matures se déroule dans un microenvironnement spécialisé, le thymus, orchestrée par des interactions complexes entre cytokines, chimiokines et cellules stromales. Les mutations bloquant la différenciation des cellules T ont un impact sur l'architecture du thymus, soulignant l’importance des interactions entre cellules T en développement et cellules stromales thymiques.Les déficits immunitaires combinés sévères sont généralement traités, avec succès, par transplantation de CSH allogénique par voie intraveineuse. Cependant, des complications peuvent survenir notamment en cas de greffe non compatible. Pour pallier à cela, la thérapie génique a été développée mais son efficacité et son innocuité restent à améliorer. Dans ce but, notre groupe a développé une approche par correction génique des progéniteurs T directement in vivo, via un vecteur lentiviral. Bien qu’efficace, là encore, l’efficacité de traitement reste insuffisante voire extrêmement limitée chez les macaques.Lors de ma thèse, j'ai donc évalué le potentiel de différents sérotypes de vecteur viraux adéno-associés (AAV) pour la transduction des thymocytes. L'administration IT de plusieurs sérotypes de AAV2 engendre une transduction des thymocytes >10 fois plus élevée que celle des vecteurs lentiviraux. Le sérotype AAV2/8 induit la transduction des thymocytes la plus efficace et les cellules transduites représentent jusqu'à 1% des cellules T périphériques d’une souris immunocompétente. En utilisant des souris immunodéficientes ZAP-70-/- comme paradigme, j'ai découvert que l'injection IT de l’AAV2/8-ZAP-70 entraîne une transduction et différentiation lymphocytaire T rapide, associée à la génération d’une medulla thymique. En effet, des cellules épithéliales thymiques de la médulla (mTEC) exprimant le régulateur auto-immun AIRE sont détectées en <2 semaines. Bien que cette reconstitution soit transitoire, les mTECs AIRE+ diminuant 10 semaines post-injection, les cellules T périphériques corrigées persistent >40 semaines et présentent environ 1 copie du vecteur AAV/cellule. Ces cellules T effectrices peuvent sécréter des niveaux élevés de cytokines et un nombre important de cellules T régulatrices est également généré. Ainsi, une seule vague de thymopoïèse à partir de progéniteurs transduits par l’AAV-ZAP-70, permet une restauration, rapide et transitoire de l'architecture thymique mais, à long terme de cellules T périphériques fonctionnelles.Pour évaluer les diverses populations de TEC régissant le développement et la sélection des cellules T, j'ai collaboré avec les groupes de P Jay/J Abramson/I Amit pour établir une cartographie de novo du compartiment stromal thymique. Nos analyses ont mis en évidence quatre populations majeures de mTEC (I-IV) avec des fonctions distinctes. Notamment, les mTEC-IV constituent une population unique présentant des similarités moléculaires et morphologiques avec les cellules tuft intestinales. Comme nous avions précédemment identifié la sécrétion d'IL-25 par les cellules tufts comme un régulateur des interactions entre compartiment épithélial et hématopoïétique dans l'intestin, nous avons évalué ce potentiel dans le thymus. Ainsi, des souris déficientes en cellules tuft intestinales présentent également une déficience spécifique en mTEC-IV et une homéostasie perturbée de diverses populations exprimant l'IL-25R dans le thymus. Notre recherche a donc permis d'identifier une nouvelle population de TEC tuft avec un rôle critique dans la formation de la niche immunitaire du thymus.L’ensemble de mes résultats montrent le potentiel thérapeutique de stratégie intrathymique de thérapie génique pour des patients ayant besoin d’une reconstitution rapide en cellules T et fournissent de nouvelles perspectives sur les populations stromales thymique et leur rôle dans l’équilibre de la niche immunitaire. / Hematopoietic stem cells (HSC) ensure the generation of all blood lineages. Their differentiation to mature T lymphocytes occurs in the specialized microenvironment of the thymus, orchestrated by complex interactions between cytokines, chemokines, and stromal cells. Mutations resulting in a block in T cell differentiation impact on the architecture of the thymus, pointing to the critical crosstalk between developing T cells and thymic stromal components.Genetic severe combined immunodeficiencies (SCID) are generally treated by the intravenous transplantation of healthy allogeneic HSCs. Although this therapy is often successful, complications can occur, especially for patients receiving non-histocompatible HSC transplants. To circumvent these problems , significant efforts have gone into developing gene therapy strategies but adverse events indicate the necessity of exploring other avenues. Our group hypothesized that in situ gene correction of T lymphoid progenitors in the thymus itself may overcome some of the drawbacks of ex vivo gene therapy. While intrathymic (IT) lentiviral vector administration corrected immunodeficient thymocyte precursors in mice, thymus transduction was inefficient and efficacy in macaques was limited.During my PhD, I assessed the in vivo potential of adeno-associated vectors (AAV) to transduce thymocyte precursors. Intrathymic administration of several different scAAV2 serotypes resulted in a >10-fold higher transduction of thymocytes (3-5%) as compared to lentiviral vectors. scAAV2/8 promoted the highest level of gene transfer and strikingly, transduced cells represented up to 1% of peripheral T lymphocytes in immunocompetent mice. Using ZAP-70-/- immunodeficient mice as a paradigm, I found that IT injection of an AAV2/8-ZAP-70 vector resulted in a rapid transduction and T cell differentiation, correlating with a dramatic generation of the thymus medulla. Indeed, medullary thymic epithelial cells (mTEC) expressing the AIRE autoimmune regulator were detected within <2 weeks. While this reconstitution was transient––AIRE+ mTECs decreased by 10 weeks post gene transfer––gene-corrected peripheral T cells, harboring approximately 1 AAV genome/ cell, persisted for >40 weeks. Effector T cells had the potential to secrete high levels of cytokines and significant numbers of gene-corrected regulatory T cells were also generated. Thus, a single wave of thymopoiesis, from intrathymic AAV-ZAP-70-transduced progenitors, allows for a rapid but transient restoration of the thymic architecture and long-term peripheral T cell function.To better assess the diverse TEC populations that orchestrate T cell development and selection, I collaborated with the groups of P. Jay/J. Abramson/I. Amit to combine single cell analysis and in-vivo fate-mapping to de novo characterize the entire stromal compartment of the thymus. Our analyses highlighted four major medullary TEC (mTEC I-IV) populations with distinct lineage regulator function and specifically, we found that mTEC-IV constitutes a highly divergent TEC subset that bears strong molecular and morphological characteristics to intestinal tuft cells. As we previously identified tuft cell secretion of IL-25 as a regulator of the crosstalk between the epithelial and hematopoietic compartments in the gut, we assessed the potential immune-modulatory function of mTEC-IV. Notably, mice deficient in intestinal tuft cells exhibited a specific depletion of mTEC-IV and a perturbed homeostasis of various IL25-R-expressing populations in the thymus. Taken together, our data identify a new tuft TEC population critical for shaping the thymus immune niche.In conclusion, the data generated during my PhD advance the therapeutic potential of intrathymic-based vector strategies for the treatment of patients requiring a rapid T cell reconstitution and provide new insights into thymic stromal subsets that are critical for shaping the thymus immune niche.