Les premières étapes de l’évolution animale restent obscures mais peuvent toutefois être appréhendées par l’étude comparative du développement des animaux basaux comme les éponges, les cnidaires ou les cténophores. Une des innovations majeures dans l’évolution des formes animales est l’apparition de l’épithélium, classiquement considérée comme une synapomorphie des eumétazoaires. Les homoscléromorphes sont les seules éponges à partager avec Eumetazoa la présence d’un véritable épithélium avec notamment une membrane basale contenant du collagène de type IV. Dans ce clade, la recherche des mécanismes épithéliaux sous-tendant le développement a pour enjeu la meilleure compréhension de leur origine et de leur importance dans l’évolution animale. Le travail de cette thèse a consisté à caractériser chez Oscarella lobularis des outils moléculaires responsables des morphogenèses épithéliales. Trois d’entre eux ont été étudiés chez l’adulte. Le premier, la voie WNT canonique, est capable d’induire l’invagination de l’épithélium externe de l’éponge, ce qui n’est pas sans rappeler ce même rôle dans d’autres contextes épithéliaux d’eumétazoaires. Le deuxième est la voie WNT non canonique ou « voie PCP », qui, quand elle est bloquée, empêche l’invagination initiée par la voie canonique. Enfin, le troisième outil est un membre de la famille des gènes à boîte T, OlTbx qui s’exprime spécifiquement dans l’épithélium après l’invagination sus-mentionnée. Cette expression rappelle des expressions d’autres gènes Tbx dans le feuillet endomésodermique invaginé lors de la gastrulation des eumétazoaires. L’invagination semble utiliser une partie d’un même programme génétique dans la gastrula des Eumetazoa et dans l’adulte des Homoscleromorpha. Mes résultats ouvrent des perspectives intéressantes concernant l’éventuelle reconnaissance d’un stade gastrula chez les éponges, point de discorde de la zoologie classique. / The first steps of animal evolution remain obscure but, nevertheless can be better understood by comparative studies of the most basally branching animals, such as sponges, cnidarians and ctenophores. Epithelium is one of the major innovations in the evolution of animal forms and is generally considered as one of the synapomorphies of Eumetazoa. The homoscleromorphs are the only sponges, with Eumetazoa, to have a true epithelium with a basal membrane containing type IV collagen. In this clade, the investigation of epithelial processes underlying development would give insights into their origin and their importance in animal evolution. The aim of my work was to characterize molecular tools involved in epithelial morphogenesis in Oscarella lobularis. I was able to characterize three of those molecular tools. The first one is the canonical WNT pathway inducing invagination of the external epithelium of the sponge, reminiscent of the same function in other epithelial contexts in Eumetazoa. The second one is the non-canonical WNT pathway or “PCP pathway” which blocks invagination when it is inhibited. The third one is a member of the T-box genes family, OlTbx, specifically expressed in the epithelial layer formed by the above-mentioned invagination. Similarly, other Tbx genes are expressed in the endomesodermal layer during eumetazoan gastrulation. Invagination processes involved in both eumetazoan gastrula and homoscleromorph adult tissue seem to share a part (WNT/Tbx) of a common genetical program. My results provide new investigation prospects, in order to answer the difficult question of the origin of gastrulation in sponges.

Doctor of Philosophy / Department of Animal Sciences and Industry / Bradley J. Johnson / Research regarding rumen epithelial adaptation and potential mechanisms during the transition period of the dairy cow is lacking. The rumen epithelium has a tremendous capacity for the absorption of volatile fatty acids (VFA) produced from microbial fermentation in the rumen. Absorption of VFA from the rumen pool delivers energy substrates to the animal and provides stability to the rumen environment. Increased epithelial surface area from the development and adaptation of rumen papillae facilitates VFA absorption. Manipulation of the diet to alter rumen fermentation can have positive effects upon the rumen papillae development supporting VFA absorption. We hypothesized that enhancing rumen epithelial surface area through dietary alterations could lead to greater VFA absorption and improve rumen stability. Experiments were conducted to determine the effects of diets formulated with cane molasses to stimulate the production of ruminal butyrate and thereby increase rumen epithelial surface area and to investigate a potential mechanism for glucagon-like peptide-2 (GLP-2) to impact epithelial development. Feeding cane molasses in the dry period improved dry matter intake during the close-up period and during lactation. Milk production was increased for cows that were fed cane molasses during the dry period. Ruminal absorption of valerate was greater during the close-up period than the far-off period but was not influenced by the addition of cane molasses. Total VFA concentration measured during the dry period was not affected by the addition of cane molasses to the diet. The presence of glucagon-like peptide receptor (GLP-2R) mRNA was confirmed in bovine tissue obtained from rumen epithelium, omasum, abomasum, duodenum, jejunum, ileum, large intestine, and pancreas. The greatest level of expression of mRNA for GLP-2R was in the small intestine and large intestine. Expression of GLP-2R mRNA during the prepartum period tended to be increased with the addition of cane molasses. Postpartum expression of GLP-2R was not increased by supplementing cane molasses in the dry cow diet. Results from these experiments indicate that dry cow diets formulated to contain cane molasses can positively influence transition cow performance and that the presence of glucagonlike peptide-2 receptor could play a pivotal role in rumen epithelial development.

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

In this thesis, I examined the expression of CD1d on respiratory epithelial cells (REC) in human and explored its potential role in mucosal immunity in the lungs. Hitherto, there have been no published reports of CD1d expression on REC though it has been observed on other epithelial surface (notably intestinal epithelial cells). This observation, and work in my supervisor’s laboratory demonstrating CD1d-restricted natural killer T cells (iNKT) cells as early players in the lungs of influenza A virus (IAV)–infected mice prompted my interest in this area. I hypothesized that CD1d is expressed on REC and that it contributes to activation of iNKT cells in the lungs via presentation of endogenous or pathogenic glycolipids. I asked following questions – i) is CD1d expressed on REC ii) can this expression be regulated and iii) does CD1d expression on REC have a function. This thesis provides the first evidence for CD1d expression on human RECs (in cell lines and primary RECs) and also presence of alternatively spliced variants. CD1d expression was inducible by viral-associated signals in vitro and despite being non-professional antigen presenting cells, RECs can present glycolipid (α–GC) to, and activate iNKT cells in a CD1d-dependent process resulting in production of both Th1 and Th2 cytokines. Using whole genome expression profiling, I then showed that iNKT cells expressed a distinct profile of genes while in direct contact with α–GC-bound CD1d on RECs compared to cells separated by transwell membrane. Here early biological pathways were dominated by cytokine and chemokine related genes (JAK-STAT signaling pathways, cytokine-responsive elements and cytokine/chemokine genes) and apoptosis-related genes. This suggested that glycolipid-bound CD1d on REC was capable of inducing a programme of immune activation in iNKT cells. I concluded my work by examining if CD1d expression on RECs influenced its active role in immunity. Using wild type and CD1d-deficient transgenic mice challenged with IAV, I showed that CD1d expression is induced on REC in vivo after viral challenge, and in the absence of CD1d, mice showed worse outcome. RECs isolated from CD1d-deficient mice had a much stronger gene expression profile for pro-inflammatory genes. This suggested that CD1d expression on REC could have a bi-directional effect – on the RECs that expressed CD1d (preventing excessive immune-related genes activation) and on the iNKT cells that it engaged (activation, with pro-immunity effects). The thesis concludes with discussion of the potential implications of these findings and future work to examine hypotheses generated from this work.

<p>Transferrin receptor 1 (Tfr1) serves as a receptor for transferrin, an iron-binding protein in the blood, in its canonical role of iron assimilation. Tfr1 is expressed ubiquitously in many tissues and is believed to be required for iron uptake by most cells. </p><p>The Tfr1 global knockout mouse highlights the requirement for Tfr1 in erythrocyte precursors. The erythron is the tissue with the highest iron requirement, to enable hemoglobin production. Tfr1-null embryos die by embryonic day 12.5 with anemia, which has been assumed to cause lethality of the knockout mice. Due to the embryonic lethality of the mice, the role of Tfr1 has not been well characterized in other tissues in vivo. This thesis examines the role of Tfr1 in other tissues through the generation and characterization of conditional knockout mouse models of Tfr1 deletion in the intestinal epithelium, pancreas, and skin.</p><p>Tfr1 is expressed on the basolateral surface of proliferating cells in the intestinal epithelium. Deletion of Tfr1 specifically in the intestinal epithelium resulted in the loss of intestinal epithelial homeostasis, loss of proliferation, lipid accumulation, gene expression indicating epithelial to mesenchymal transition of intestinal epithelial cells, and early neonatal lethality. These phenotypes were mostly alleviated by forced expression of a mutant Tfr1 allele which is unable to bind to iron-loaded transferrin, suggesting that Tfr1 has a novel role independent of its canonical iron-assimilatory ability.</p><p>Deletion of Tfr1 in the pancreas resulted in juvenile death due to perturbed homeostasis of both endocrine and exocrine tissues, resulting in symptoms associated with pancreatitis and diabetes. No diabetic phenotype was detected in the conditional knockout mouse model of Tfr1 deletion specifically in &#946;-cells, suggesting that the primary effect of the loss of Tfr1 was limited to the exocrine tissue.</p><p>Deletion of Tfr1 in the epidermis of the skin caused neonatal lethality with abnormal hair follicle morphology and a significant reduction in dermal adipocytes.</p><p>These results indicate that the loss of Tfr1 has pleiotropic effects, depending on the cell type affected. Furthermore, Tfr1 appears to have non-canonical functions in the intestinal epithelium, a novel discovery.</p> / Dissertation

Nuclear Factor kappaB (NF-êB) is a transcription factor whose activation is increased in settings of allergic asthma. At least two parallel NF-êB pathways exist: the classical pathway, which plays a role in inflammation and cell survival, and the alternative pathway, which regulates lymphoid cell development and organogenesis. The classical NF-êB pathway regulates inflammatory responses derived from lung epithelial cells; however, the role of the alternative pathway in lung epithelial cells remains unclear. We demonstrate that both classical and alternative NF-êB are activated in lung epithelial cells in response to multiple pro-inflammatory agonists, and siRNA-mediated knockdown of alternative NF-êB proteins largely attenuates pro-inflammatory cytokine production. Furthermore, simultaneous activation of both pathways leads to cooperative increases in pro-inflammatory responses, indicating a potential role for both classical and alternative NF-êB in the regulation of epithelial-derived pro-inflammatory responses. NF-êB activation in the epithelium modulates allergic inflammation in mouse models of allergic airway disease, however, its role in the context of an allergen relevant to human asthma remains unknown. In order to address the impact of inhibition of NF-êB in the epithelium in vivo, we utilized a House Dust Mite (HDM)-induced model of allergic airway disease. We demonstrate that HDM exposure activates classical and alternative NF-êB in both murine lung epithelium and human bronchial epithelial cells. Furthermore, following exposure to HDM, airway hyperresponsiveness, neutrophilic inflammation, and remodeling are attenuated in transgenic CC10-NF-êBSR (airway epithelial specific inhibitor of classical and alternative NF-êB) mice in comparison to wild type mice. Our data also demonstrate that specific knockdown of the alternative NF-êB protein, RelB, in the lung partially protects against HDM-induced pro-inflammatory responses, indicating that both classical and alternative NF-êB are important in HDM-induced responses. NF-êB proteins are modified by the redox-dependent post-translational modification, S-glutathionylation, under conditions of oxidative stress. S-glutathionylation of IKKâ, an upstream kinase in the NF-êB pathway, is known to decrease its catalytic activity; however, it is unknown how S-glutathionylation of IKKâ occurs. GSTP is an enzyme that catalyzes protein S-glutathionylation under conditions of oxidative stress and has been associated with the development of allergic asthma. We aimed to determine whether GSTP regulates NF-êB signaling, S-glutathionylation of IKKâ, and pro-inflammatory cytokine production. We demonstrate that siRNA-mediated knockdown of GSTP modulates NF-êB activation, NF-êB transcriptional activity, and pro-inflammatory cytokine production in response to LPS, a component of a bacterial cell wall. Furthermore, we demonstrate that GSTP associates with IKKâ in response to agonist stimulation and dampens IKKâ-induced pro-inflammatory cytokine production, surprisingly, independent of its catalytic activity. We also show that GSTP associates with other proteins of the NF-êB pathway, indicating a potential dual mechanism for repression of NF-êB-induced signaling. These studies collectively demonstrate that classical and alternative NF-êB contribute to epithelial-derived inflammatory responses, and GSTP may be a novel target by which NF-êB can be regulated.

by Vincent, Wai-ip Law. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 128-134). / Abstract also in Chinese. / Chapter Chapter I. --- Literature Review / Chapter I.1. --- "Structure, functions and general physiology of equine sweat gland" / Chapter I.1.1. --- Ultrastructure of equine sweat gland --- p.1 / Chapter I.1.2. --- Functions and physiology of equine sweat gland --- p.3 / Chapter I.1.3. --- Experimental studies on equine sweat gland by functional approaches --- p.4 / Chapter I.1.4. --- Hormonal and neuronal regulation of sweat secretion in equidaes --- p.5 / Chapter I.1.5. --- Possible role(s) of extracellular ATP in equine sweat gland epithelia --- p.6 / Chapter I.1.6. --- Measurement of electrogenic anion secretion by short-circuit current (Isc) technique --- p.8 / Chapter I.2. --- Classification of purinergic receptors and its existence in biological systems / Chapter I.2.1. --- Functional classification of purinergic receptors --- p.13 / Chapter I.2.2. --- Basic structure of G-protein coupled P2Y receptors --- p.17 / Chapter I.2.3. --- Physiological function and significance of purinergic receptors --- p.20 / Chapter I.3. --- Objectives of study --- p.22 / Chapter Chapter II. --- Methods and Materials / Chapter II.l. --- Culture technique of the equine epithelial cells --- p.23 / Chapter II.2. --- Conventional short-circuit current (Isc) measurement technique / Chapter II.2.1. --- Introduction --- p.25 / Chapter II.2.2. --- Preparation of permeable supports and electrodes --- p.25 / Chapter II.2.3. --- Experimental set up and measurement of Isc --- p.28 / Chapter II.2.4. --- Measurement of Isc during experiment --- p.30 / Chapter II.3. --- Measurement of intracellular free calcium ( [Ca2+ ]ii) by microspectrofluorimetry / Chapter II.3.1. --- Preparation of cells --- p.31 / Chapter II.3.2. --- The set up and procedures for experiment --- p.31 / Chapter II.4. --- Simultaneous measurement of changes in [Ca2+ ]i and Isc / Chapter II.4.1. --- Experimental set up and manipulation --- p.35 / Chapter II.4.2. --- Other preparations before experiment --- p.37 / Chapter II.5. --- Material and solutions used for experiment / Chapter II.5.1. --- Culture media and enzyme --- p.40 / Chapter II.5.2. --- Chemicals and Drugs --- p.40 / Chapter II.5.3. --- Preparation of solution for experiments --- p.42 / Chapter II.6. --- Statistical analysis --- p.44 / Chapter Chapter III. --- Results / Chapter III.l. --- Effects of nucleotides on transepithelial ion transport / Chapter III.1.1. --- Basic electrophysiological properties of cultured equine sweat gland epithelia --- p.45 / Chapter III. 1.2. --- Short-circuit current (Isc) induced by nucleotides --- p.45 / Chapter III. 1.3. --- Identification of ion species responsible for the change in Isc --- p.50 / Chapter III. 1.4. --- Effects of chloride channels blockers on the UTP-induced Isc --- p.51 / Chapter III.2. --- Signal transduction mechanisms of P2Y-nucleotide receptors / Chapter III.2.1. --- The involvement of Gi-proteins --- p.56 / Chapter III.2.2. --- Effect of BAPTA on the increases in Isc induced by nucleotides --- p.58 / Chapter III.2.3. --- Study of P2Y-receptor mediated increase in [Ca2+]i --- p.62 / Chapter III.3. --- Characterization of the P2Y subtype(s) by cross desensitization experiments / Chapter III.3.1. --- Autologous desensitization experiments --- p.70 / Chapter III.3.2. --- Classical cross desensitization experiments --- p.70 / Chapter III.3.3. --- Characterization of the ATP-insensitive P2Y-receptor --- p.80 / Chapter III.3.4. --- Interaction between ATP and bradykinin --- p.87 / Chapter III.4. --- Simultaneous measurement of [Ca2+]i and Issc / Chapter III.4.1. --- Effect ofUDP and ADP --- p.89 / Chapter III.4.2. --- Correlation of Isc and [Ca2+]i --- p.92 / Chapter III.4.3. --- Cross desensitization experiments --- p.97 / Chapter III.5. --- Evidence of a [Ca2+]i-independent Isc-component induced by nucleotides / Chapter III.5.1. --- The time course of the ΔRf and ΔISC --- p.102 / Chapter III.5.2. --- Effect of ionomycin on the ΔISC and ΔRf induced by nucleotides --- p.110 / Chapter III.5.3. --- Effect of thapsigargin on the ΔISC and ΔRf induced by nucleotides --- p.110 / Chapter III.5.4. --- Effect of thapsigargin in nominal Ca2+-free solution --- p.115 / Chapter Chapter IV. --- Discussion / Chapter IV. 1. --- Role of extracellular nucleotides in epithelial tissues --- p.119 / Chapter IV.2. --- Characterization of an ATP-insensitive P2Y-nucleotide receptor --- p.120 / Chapter IV.3. --- Expression of the novel ATP-insensitive receptor on a functionally polarized epithelia --- p.122 / Chapter IV.4. --- Involvement of a [Ca2+]i -independent Isc induced by nucleotides --- p.124 / Chapter Chapter V. --- References --- p.128

INTRODUCTION: The cornea forms the anterior-most barrier of the eye, consisting of a non-keratinized pseudostratified squamous epithelium, a collagen-based stroma, and an endothelium. It is completely avascular, yet the most densely innervated structure in the human body. The sensory nerves project from the ophthalmic branch of the trigeminal cranial nerve into the limbal/stromal interface. From there, the nerves branch and ascend into Bowman’s membrane, a basal lamina delineating the epithelium from the stroma, and project into the epithelium as free nerve endings. Injury to the corneal epithelium can potentially lead to impaired vision if the wound healing process is not properly initiated. Immediately after injury, nucleotides such as ATP are released and bind to purinergic receptors known to be located in epithelial cell membranes, thereby initiating epithelial cell migration to close the wound. Malfunctions in the interactions between the corneal nerves and their epithelial counterparts during the wound healing process are thought to contribute to the attenuated wound healing characteristic of diabetes. However, the precise nature of these interactions, how they facilitate wound healing, and how they are impaired in diabetes, is not well understood. OBJECTIVES: Previously, our lab has shown that a member of purinergic family receptors (P2X7) is localized in the basal epithelial cells and becomes relocated to the leading edge of the wound after injury. When the relocation is inhibited, migration is attenuated. Additionally, it is known that diabetic mouse models display slower wound healing rates. The present study has three aims: (1) to replicate the characteristic sub-basal whorl organization of the corneal nerves in organ-cultured corneas; (2) to elucidate the connections between patterns of corneal innervation and purinergic receptor expression; and (3) to understand how these patterns interact to facilitate normal wound healing and how these interactions are disrupted in a diabetic model. METHODS: Our approach was to use immunohistochemistry of dissected mouse and to visualize the tissue using confocal microscopy. Sensory innervation profiles from diet induced obesity (DIO) mouse corneas and their wildtype C57Bl6 counterparts were compared in unwounded and wounded tissue. To image the nerves a methanol fixation protocol was optimized to examine the sub-basal plexus and the apical nerves. Corneas were dissected, stained with beta III-tubulin, which identifies nerves, and with an antibody to the P2X7 purinergic receptor, which is expressed in the epithelium and nerves. Trephine induced epithelial abrasion injuries were made on separate DIO and control models to compare re-epithelialization and re-innervation between the diseased and healthy states. Corneas were imaged using a Zeiss LSM 700 laser scanning confocal microscope and optical images were taken through the cornea over a distance averaging 115 microns. Corneas were imaged using a macro tiling plugin, stitching 3x3 optical z-stacks into composite images. The 3x3 tiles were created to image the central whorl, as well as the peripheral nerve fibers. Co-localization of P2X7 and betaIII tubulin were determined by thresholding using ImageJ/FIJI software. RESULTS: The elegant organization of the centralized sub-basal whorl of the control mouse was disrupted in the DIO mouse cornea, appearing fragmented and incomplete. Analysis of 7.5 and 15 wk corneas showed the whorl to be present at 7.5 wks. Average apical nerve fiber projection length was decreased in DIO cornea. Yet, analyses at each epithelial layer demonstrated overall increased apical nerve density in the DIO corneas as compared to control while sub-basal nerve density decreased dramatically. Stromal nerves remained equivalent. P2X7 did co-localize to the large stromal nerve fibers but it was difficult to show the localization along the sub-basal nerve plexus. However in cross-section images, P2X7 displayed an intracellular polarity, and was present along the apical surface of the columnar basal epithelial cells lining the basement membrane. This localization may suggest the presence of P2X7 expressing sensory nerves, which may be ideally poised for communication with the basal cells after injury. CONCLUSIONS: These data support the hypothesis that there is indeed a difference between diabetic and control corneal innervation. While wound healing differences due to the interaction between sensory nerves and the localization of P2X7 in epithelium at the leading edge remain to be fully elucidated, the novel finding of P2X7 expression in corneal nerves confirms a potential role of purinergic receptor and nerve coordination in conducting the wound healing response.

No description available.

De par son rôle dans de nombreuses fonctions biologiques, la vitamine A est une molécule majeure et cruciale du développement embryonnaire à l’âge adulte. Celle-ci est aussi, à l’heure actuelle, déjà largement utilisée comme agent thérapeutique pour de nombreuses pathologies affectant principalement la peau et les yeux (cancer, acné, psoriasis,brûlures oculaires) et certains cancers. Son action pro-cicatrisante - bien que largement étudiée grâce à de nombreux modèles humains et animaux - reste encore aujourd’hui mal caractérisée quant aux mécanismes d’action moléculaire (régulation de gènes) et cellulaire(migration, prolifération…) qu’elle utilise.Dans le but d’améliorer et de mieux maîtriser l’utilisation de la vitamine A dans le traitement de lésions telles que les brûlures oculaires, ce travail visait d’une part, à étudier, plus en détail,l’effet de l’acide tout-trans rétinoïque ou ATRA - dérivé le plus actif - sur la cicatrisation de ’épithélium cornéen en utilisant un modèle in vivo de brûlures cornéennes par base chez la souris. Son but était également de déterminer par quels processus cellulaires, l’ATRA agit, en utilisant cette fois-ci un modèle in vitro (cellules épithéliales cornéennes humaines). Enfin,nous nous sommes intéressés à la régulation de gènes cibles par l’ATRA au niveau de la sphère oculaire, connus pour être impliqués dans la dynamique de la MEC.Ainsi, nous avons démontré que l’ATRA permettait la cicatrisation de l’épithélium cornéen en agissant principalement sur la migration cellulaire. Puis nous avons identifié un gène :LOXL4 - membre de la famille des lysyl oxydases - dont l’expression est induite par l’ATRA,et nous avons prouvé que son rôle était essentiel dans la cicatrisation cornéenne, décrivant ainsi pour la première fois un lien génique direct et protéique entre la vitamine A, substance active et son action clinique pro-cicatrisante. / Because of its role in many biological functions, Vitamin A is a major and crucialmolecule from development to adulthood. Currently, it is largely used as therapeutic agent inseveral eye or skin pathologies (acne, psoriasis, ocular burns) and cancers. Its pro-healingproperties have been largely studied in human and animal models but molecular (generegulations) and cellular (migration, proliferation…) mechanisms of the vitamin A actionhave to be more detailed.In order to better improve and control its use in the treatment of lesions such as ocular burns,this work aimed to study, more in details, the effects of atRA (all-trans-retinoic acid), activederivative form of vitamin A, on corneal epithelium healing using an in vivo model of alkaliocular burns in mouse and then, to determine by which cellular process atRA acts, by usingthis time, an in vitro model (human corneal epithelial cells). Finally, we were interested intargeting genes regulation by atRA in the ocular sphere, specially known to be implied in theECM dynamic.First, we demonstrated that atRA improves corneal epithelium wound healing, essentially byacting on migration. Then, we identified a gene, LOXL4, member of the lysyl oxidase family,which expression is induced by atRA and we proved that this one is essential in cornealwound healing, describing for the first time a direct gene and protein link between vitamin A,active substance, and its clinical pro-healing action.